Guidance on the Biocidal Products Regulation . Volume II, Efficacy. Assessment and evaluation (Parts B+C).
Date: 2021-12-31
Institutions: European Chemicals Agency
Subjects: biochemistry, chemical product, plant health product, regulation (EU), toxic substance, user guide

G U I D A N C E  

Guidance on the Biocidal Products Regulation 

Volume II Efficacy - Assessment and Evaluation (Parts B+C) 

Version 4.0 
December 2021 

 
 
 
 
 
 
 
Guidance on the BPR: Volume II Parts B+C  
Version 4.0 December 2021 

2 

LEGAL NOTICE 

This document aims to assist users in complying with their obligations under the Biocidal 
Products  Regulation  (BPR).  However,  users  are  reminded  that  the  text  of  the  BPR  is  the 
only  authentic  legal  reference  and  that  the  information  in  this  document  does  not 
constitute legal advice. Usage of the information remains under the sole responsibility of 
the user. The European Chemicals Agency does not accept any liability with regard to the 
use that may be made of the information contained in this document. 

Guidance  on  the  BPR:  Volume  II  Efficacy  -  Assessment  and  Evaluation  (Parts 
B+C) 

Reference:  
Cat. Number:  
ISBN:  
DoI:  
Publ.date:  
Language:  

ECHA-21-H-11-EN 
ED-07-21-104-EN-N 
978-92-9468-017-4 
10.2823/796364 
December 2021 
EN 

© European Chemicals Agency, 2021 

If  you  have  questions  or  comments  in  relation  to  this  document  please  send  them 
(quote  the  reference  and  issue  date)  using  the  information  request  form.  The 
information  request  form  can  be  accessed  via  the  Contact  ECHA  page  at: 
http://echa.europa.eu/contact.  

European Chemicals Agency 

Mailing address: P.O. Box 400, FI-00121 Helsinki, Finland 
Visiting address: Telakkakatu 6, FI-00150 Helsinki, Finland 

 
 
 
 
 
   
 
 
 
 
Guidance on the BPR: Volume II Parts B+C  
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DOCUMENT HISTORY 

Version 

Comment 

Version 1.0  First edition 

Version 2.0  Update to PT8 

Date 

February 2017 

December 2017 

The text has been revised as follows: 

•  To add a new Appendix 12;  

•  To  revise  section  5.5.8.3  to  remove  temporary 

footnote re. the new Appendix 12; 

•  To  add  a  new  footnote  to  section  5.5.8.2.2.3,  Use 

Class 2, Test Species; 

•  To re-number Appendices after the new Appendix 12 

and revise all cross references to these Appendices.  

Version 3.0  Update to PT5 Drinking Water Disinfectants: 

April 2018 

•  Section 5.4.5 has been revised and updated; 

•  Appendix 3 has been revised for PT5; 

•  Appendix 4 has been revised for PT5. 

In  addition  corrections  and  editorial  revisions  have  been 
made as follows:  

•  Section,  5.5.8.2.2.3:  the  footnote  added  in  version 
2.0,  (footnote  28)  has  been  moved  to  the  beginning 
of  the  section  because  it  applies  to  Use  Class  1  as 
well as Use Class 2. It is now footnote 24; 

•  Appendix 3: corrections are made to: 

o  Escherichia  coli  ATCC 10536: amend O to (X) 

for PT2 and PT3; 

o  Escherichia coli K12 NCTC 10538: amend X to 

(X) for PT2;  

o  Addition  of  Proteus  vulgaris  ATCC  13315  (not 

for teat disinfection) for PT3. 

•  Appendix  4:  the  tables  that  were  on  the  ECHA 
Biocides  webpages,  have  been  moved  into  the 
Guidance  document  because  the  revised  information 
in  the  tables  is  directly  linked  and  reflected  in  the 
guidance  text  and  the  “Notes”  are  giving  guidance 
not simply test methodology details; 

•  Appendix 4: corrections are made to: 

o  Corrections 

to  Guidance 

section 

cross 

 
   
 
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Guidance on the BPR: Volume II Parts B+C  
Version 4.0 December 2021 

references for PT4 - surfaces in drinking water 
systems.  

o  Correction of loq reduction in EN 13623 to 4 

•  Appendix  16:  a  note  has  been  added  to  inform 
readers  that  this  appendix  contain  information  that 
refers  to  the  BPD  and  which  is  now  obsolete  and 
should also refer to the text in sections 2 and 5; this 
will be revised at the next update. 

Version 4.0  Update to: 

December 2021 

List of Abbreviations. 

PT18  -  Section  5.6.4  -  Insecticide,  Acaricides  &  other 
Biocidal  Products  against  Arthropods+  PT  19  Repellents  & 
Attractants (arthropods): 

•  The 

title  has  been  amended 

to 

Insecticides, 

acaricides and products to control other arthropods; 

•  All  information  related  to  attractants  and  repellents 

has been removed from section 5.6.4; 

PT19  -  Section  5.6.5  PT19  Repellents  and  attractants  has 
been added; 

Appendix 17: Table 40: PT 19 – Repellents & Attractants has 
been removed. 

Appendix 18: has been updated with relation to PT19. 

 
 
 
 
 
   
 
 
Guidance on the BPR: Volume II Parts B+C  
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5 

PREFACE  

The  Guidance on the Biocidal Products Regulation (BPR) is to be applied to applications for 
active substance approval and product authorisation as submitted from 1 September 2013, 
the date of application (DoA) of the Biocidal Product Regulation (the BPR). 

This document describes the BPR obligations and how to fulfil them. 

The  scientific  guidance  provides  technical  scientific  advice  on  how  to  fulfil  the  information 
requirements set by the BPR (Part A) and how to asses and evaluate the efficacy to establish 
the  benefit  arising  from  the  use  of  biocidal  products  and  to  prove  that  it  is  sufficiently 
effective (Parts B+C). 

In  addition  to  the  BPR  guidance,  the  Biocidal  Products  Directive  (BPD)  guidance  and  other 
related documents are still considered applicable for new submissions under the BPR  in the 
areas  where  there  are  no  BPR  guidance  or  it  is  under  preparation.  Furthermore  these 
documents  are  still  valid  in  relation  to  the  applications  for  active  substance  approval 
submitted  under  the  BPD  that  may  still  be  under  evaluation.  Also  the  Commission  has 
addressed  some  of  the  obligations  in  further  detail  in  the  Biocides  Competent  Authorities 
meetings  documents  which  applicants  are  advised  to  consult.  Please  see  ECHA  Biocides 
Guidance  website 
links  to  these  documents:  [https://echa.europa.eu/guidance-
for 
documents/guidance-on-biocides-legislation]. 

Applicability of Guidance 

Guidance  on  applicability  of  new  guidance  or  guidance  related  documents  for  active 
substance  approval  is  given  in  the  published  document  “Applicability  time  of  new  guidance 
and  guidance-related  documents  in  active  substance  approval”  available  on  the  BPC 
Webpage1  and  for  applicability  of  guidance  for  product  authorisation,  please  see  the  CA-
document  CA-july2012-doc6.2d 
the  ECHA  Guidance  page 
[https://echa.europa.eu/documents/10162/23036409/ca-july12-doc_6_2d_final_en.pdf]. 

(final),  available  on 

1  Link  available  under  Working  Procedures  (right  column)  [https://echa.europa.eu/about-us/who-we-are/biocidal-
products-committee] 

 
   
 
 
 
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Table of Contents 

LEGAL NOTICE .................................................................................................. 2 

DOCUMENT HISTORY ......................................................................................... 3 

PREFACE .......................................................................................................... 5 

LIST OF ABBREVIATIONS ................................................................................... 19 

DIRECTORATE-GENERAL HEALTH AND CONSUMER PROTECTION ............................. 20 

NEW NAME: DIRECTORATE-GENERAL FOR HEALTH AND FOOD SAFETY (SANTE) ....... 20 

GLOSSARY OF TERMS ........................................................................................ 22 

1. GENERAL INTRODUCTION ............................................................................... 26 

2. CLAIMS ........................................................................................................ 26 
2.1 INTRODUCTION ....................................................................................... 26 
2.2 LABEL CLAIMS AND DIRECTIONS FOR USE .................................................. 27 

3. GENERAL CONSIDERATIONS FOR THE DEVELOPMENT AND REPORTING OF EFFICACY 
DATA 28 

3.1 EFFICACY ................................................................................................ 28 
3.1.1 Efficacy tests .................................................................................. 28 
3.1.2 Test report ..................................................................................... 29 
3.2 RESISTANCE ........................................................................................... 30 

4. ACTIVE SUBSTANCE APPROVAL ....................................................................... 30 
4.1 INTRODUCTION ....................................................................................... 30 
4.2 GENERAL PRINCIPLES .............................................................................. 31 
4.2.1 Intended use .................................................................................. 31 
4.2.2 Evaluation of efficacy ....................................................................... 31 
4.2.2.1 Active substance efficacy (part A): .......................................... 31 
4.2.2.2 Product efficacy (part B): ....................................................... 33 
4.2.3 Overall evaluation for active substance approval ................................. 33 
4.2.4 Link to risk assessment .................................................................... 33 
4.3 ACTIVE SUBSTANCES WHICH ARE NOT INTENDED TO BE USED IN ISOLATION 34 
4.4 “DUMMY PRODUCTS” ................................................................................ 35 
4.5 ACTIVE SUBSTANCES USED IN TREATED MATERIALS AND TREATED ARTICLES 35 
4.5.1 Efficacy assessment for active substance approval ............................... 35 
4.5.2 Efficacy assessment for active substances in specific PTs ...................... 35 

5. PRODUCT AUTHORISATION............................................................................. 37 
5.1 EVALUATION OF EFFICACY AT PRODUCT AUTHORISATION STAGE ................. 37 
5.2 PRODUCT FAMILIES ................................................................................. 37 
5.2.1 Background .................................................................................... 37 
5.2.2 Worst case testing ........................................................................... 38 
5.2.3 Take formulation types and chemical composition into account ............. 41 
5.2.4 Allowing for the addition of new products in a family ............................ 42 
5.2.5 Deviation in meta SPC’s ................................................................... 43 
5.2.6 Minimum concentration needed ......................................................... 44 
5.3 TREATED ARTICLES .................................................................................. 44 

 
 
 
 
 
   
 
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5.3.1  The  basic  distinction  between  material  protection  and  protection  of 

humans or animals ........................................................................ 46 
5.4 DISINFECTANTS (MAIN GROUP 1) .............................................................. 48 
5.4.0 General .......................................................................................... 48 
5.4.0.1 Introduction ......................................................................... 48 
5.4.0.2 Dossier requirements ............................................................ 49 
5.4.0.3 Label claim .......................................................................... 49 
5.4.0.3.1 Target Organisms .................................................... 50 
5.4.0.3.2 Areas of Use ............................................................ 51 
5.4.0.3.3 Sites of Application ................................................... 51 
5.4.0.3.4 Directions for use (Methods of application) .................. 52 
5.4.0.3.5 Other interfering parameters ..................................... 52 
5.4.0.4 Efficacy testing ..................................................................... 52 
5.4.0.4.1 Tiered approach ....................................................... 52 
5.4.0.4.2 Standard test methods ............................................. 54 
5.4.0.4.3 Data requirements ................................................... 56 
5.4.0.4.4 Relevant factors of the test procedure ........................ 58 
5.4.0.5 General data requirements .................................................... 62 
5.4.0.5.1 Test range............................................................... 62 
5.4.0.5.2 Claim for several areas of use .................................... 62 
5.4.0.5.3 Biocidal products with biostatic effect ......................... 62 
5.4.0.5.4 Malodour control ...................................................... 62 
5.4.0.5.5 Changes in ingredients  ............................................ 62 
5.4.0.5.6 Treated articles ........................................................ 62 
5.4.0.5.7 Biocidal Product Families ........................................... 62 
5.4.0.6 Resistance ........................................................................... 62 
5.4.0.7 Assessment of application for authorisation .............................. 63 
5.4.0.7.1 Decision making ...................................................... 63 
5.4.0.7.2 Assessment ............................................................. 63 
5.4.1 PT1 Human hygiene biocidal products ................................................ 63 
5.4.1.1 Introduction ......................................................................... 63 
5.4.1.2 Hand disinfectants ................................................................ 64 
5.4.1.2.1 Introduction ............................................................ 64 
5.4.1.2.2 Data requirements ................................................... 64 
5.4.1.2.3 Acceptance criteria ................................................... 66 
5.4.1.3 Other skin and scalp disinfection ............................................. 66 
5.4.1.3.1 Data requirements ................................................... 66 
5.4.1.3.2 Acceptance criteria ................................................... 67 

5.4.2  PT2  Disinfectants  and  algaecides  not  intended  for  direct  application  to 

humans or animals ........................................................................ 68 
5.4.2.1 Introduction ......................................................................... 68 
5.4.2.2 Data requirements ................................................................ 68 
5.4.2.2.1 Use in health care .................................................... 69 
5.4.2.2.2 Tuberculosis departments ......................................... 69 
5.4.2.2.3 Cleanrooms ............................................................. 69 
5.4.2.2.4 Products against viruses ........................................... 69 
5.4.2.3 Disinfectants for hard surfaces (in PT2) ................................... 69 
5.4.2.3.1 Introduction ............................................................ 69 
5.4.2.3.2 Data requirements ................................................... 70 
5.4.2.3.3 Acceptance criteria ................................................... 71 
5.4.2.4 Soft furnishings .................................................................... 71 

 
   
 
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5.4.2.4.1 Introduction ............................................................ 71 
5.4.2.4.2 Data requirements ................................................... 71 
5.4.2.4.3 Acceptance criteria ................................................... 72 
5.4.2.5 Room disinfection with vaporised biocide ................................. 72 
5.4.2.5.1 Introduction ............................................................ 72 
5.4.2.5.2 Data requirements ................................................... 74 
5.4.2.5.3 Acceptance criteria ................................................... 75 
5.4.2.5.4 Notes ..................................................................... 76 
5.4.2.6 Swimming pools, spas and hot tubs ........................................ 76 
5.4.2.6.1 Introduction ............................................................ 76 
5.4.2.6.2 Data requirements ................................................... 77 
5.4.2.6.3 Acceptance criteria ................................................... 77 
5.4.2.7 Toilets ................................................................................. 78 
5.4.2.7.1 Introduction ............................................................ 78 
5.4.2.7.2 Data requirements ................................................... 78 
5.4.2.7.3 Acceptance criteria ................................................... 79 
5.4.2.8 Air-conditioning systems ........................................................ 79 
5.4.2.8.1 Introduction ............................................................ 79 
5.4.2.8.2 Data requirements ................................................... 80 
5.4.2.8.3 Acceptance criteria ................................................... 80 
5.4.2.9 Equipment disinfection by immersion ...................................... 80 
5.4.2.9.1 Introduction ............................................................ 80 
5.4.2.9.2 Data requirements ................................................... 81 
5.4.2.9.3 Acceptance criteria ................................................... 81 
5.4.2.10 Textiles.............................................................................. 82 
5.4.2.10.1 Introduction........................................................... 82 
5.4.2.10.2 Data requirements ................................................. 82 
5.4.2.10.3 Acceptance criteria ................................................. 84 
5.4.2.11 Biofilms ............................................................................. 84 
5.4.2.11.1 Introduction........................................................... 84 
5.4.2.11.2 Data requirements ................................................. 85 
5.4.2.11.3 Acceptance criteria ................................................. 88 
5.4.2.12 Soil ................................................................................... 89 
5.4.2.13 Other uses in PT2 ............................................................... 89 
5.4.3 PT3 Veterinary hygiene biocidal products ............................................ 89 
5.4.3.1 Introduction ......................................................................... 89 
5.4.3.2 Disinfectants for hard surfaces in PT3 ...................................... 90 
5.4.3.2.1 Introduction ............................................................ 90 
5.4.3.2.2 Data requirements ................................................... 90 
5.4.3.2.3 Acceptance criteria ................................................... 92 
5.4.3.3 Disinfection of bee hives and beekeeping equipment ................. 92 
5.4.3.3.1 Introduction ............................................................ 92 
5.4.3.3.2 Data requirements ................................................... 93 
5.4.3.3.3 Acceptance criteria ................................................... 94 
5.4.3.4 Animal feet disinfection ......................................................... 94 
5.4.3.4.1 Introduction ............................................................ 94 
5.4.3.4.2 Data requirements ................................................... 94 
5.4.3.4.3 Acceptance criteria ................................................... 95 
5.4.3.5 Teat disinfection ................................................................... 96 
5.4.3.5.1 Introduction ............................................................ 96 
5.4.3.5.2 Data requirements ................................................... 96 

 
 
 
 
 
   
 
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5.4.3.5.3 Acceptance criteria ................................................... 98 
5.4.3.6 Other animal corporal hygiene ................................................ 98 
5.4.3.6.1 Introduction ............................................................ 98 
5.4.3.6.2 Data requirements ................................................... 99 
5.4.3.6.3 Acceptance criteria ................................................... 100 
5.4.3.7 Disinfection of hatching-eggs ................................................. 100 
5.4.3.7.1 Introduction ............................................................ 100 
5.4.3.7.2 Data requirements ................................................... 100 
5.4.3.7.3 Acceptance criteria ................................................... 102 
5.4.3.8 Textile disinfection in PT3 ...................................................... 102 
5.4.3.8.1 Introduction ............................................................ 102 
5.4.3.8.2 Data requirements ................................................... 102 
5.4.3.8.3 Acceptance criteria ................................................... 103 

5.4.3.9 Disinfection of manure, litter and other substrates for veterinary 

use ...................................................................................... 103 
5.4.3.9.1 Introduction ............................................................ 103 
5.4.3.9.2 Data requirements ................................................... 104 
5.4.3.9.3 Acceptance criteria ................................................... 105 
5.4.3.10 Other uses in PT3 ............................................................... 105 
5.4.4 PT4 Food and feed area disinfectants ................................................. 105 
5.4.4.1 Introduction ......................................................................... 105 
5.4.4.2 Disinfection of hard surfaces in food and feed area PT4 ............. 106 
5.4.4.2.1 Introduction ............................................................ 106 
5.4.4.2.2 Data requirements ................................................... 106 
5.4.4.2.3 Acceptance criteria ................................................... 108 
5.4.4.3 Disinfection of inner surfaces in PT4 ........................................ 108 
5.4.4.3.1 Introduction ............................................................ 108 
5.4.4.3.2 Data requirements ................................................... 108 
5.4.4.3.3 Acceptance criteria ................................................... 110 
5.4.4.4 Equipment disinfection by soaking .......................................... 110 
5.4.4.4.1 Introduction ............................................................ 110 
5.4.4.4.2 Data requirements ................................................... 110 
5.4.4.4.3 Acceptance criteria ................................................... 112 
5.4.4.5 Disinfection in dish washing machines and crate washers .......... 112 
5.4.4.5.1 Introduction ............................................................ 112 
5.4.4.5.2 Data requirements ................................................... 112 
5.4.4.5.3 Acceptance criteria ................................................... 114 
5.4.4.6 Disinfection of inner surfaces in human drinking water systems .. 115 
5.4.4.6.1 Introduction ............................................................ 115 
5.4.4.6.2 Data requirements ................................................... 115 
5.4.4.6.3 Acceptance criteria ................................................... 117 
5.4.4.7 Disinfection of inner surfaces in veterinary water systems ......... 117 
5.4.4.7.1 Introduction ............................................................ 117 
5.4.4.7.2 Data requirements ................................................... 117 
5.4.4.7.3 Acceptance criteria ................................................... 118 
5.4.4.8 Other uses in PT4 ................................................................. 118 
5.4.5 PT5 Drinking water disinfectants ....................................................... 118 
5.4.5.1 Introduction ......................................................................... 118 
5.4.5.2 Disinfection at the drinking water suppliers and their water 

distribution systems ............................................................... 119 
5.4.5.2.1 Introduction ............................................................ 119 

 
   
 
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5.4.5.2.2 Data requirements ................................................... 120 
5.4.5.3 Disinfection of raw water for individual supply (1-2 premises) .... 121 
5.4.5.3.1 Introduction ............................................................ 121 
5.4.5.3.2 Data requirements ................................................... 121 
5.4.5.4 Disinfection in collective drinking water systems ....................... 123 
5.4.5.4.1 Introduction ............................................................ 123 
5.4.5.4.2 Data requirements ................................................... 123 
5.4.5.5 Disinfection of water in reservoirs ........................................... 127 
5.4.5.5.1 Introduction ............................................................ 127 
5.4.5.5.2 Data requirements ................................................... 127 
5.4.5.6 Disinfection of water of undefined quality for small scale use (up to 5 
L/person/day) ....................................................................... 128 
5.4.5.6.1 Introduction ............................................................ 128 
5.4.5.6.2 Data requirements ................................................... 129 
5.4.5.7 Disinfection of water for animals ............................................. 130 
5.4.5.7.1 Introduction ............................................................ 130 
5.4.5.7.2 Data requirements ................................................... 130 
5.4.6 Materials and Articles Treated to Protect Humans or Animals ................ 131 
5.4.6.1 Determining the purpose of the Treatment............................... 132 
5.4.6.2 Effects Intended to Inhibit Microbial Growth ............................. 132 
5.4.6.3 Effects intended to Kill Micro-organisms through Contact ........... 133 
5.4.6.4 Acceptance Criteria ............................................................... 134 
5.5 PRESERVATIVES (MAIN GROUP 2).............................................................. 140 
5.5.1 Distinction between preservation/curative treatment and disinfection .... 140 
5.5.1.1 Curative uses ....................................................................... 141 
5.5.1.2 Borderline case: Algaecides .................................................... 142 
5.5.1.3 Borderline cases: Treated articles ........................................... 142 
5.5.2 Principles for testing preservatives .................................................... 142 
5.5.3 Tiered approach to testing preservatives ............................................ 143 
5.5.4 Standard Test Methods .................................................................... 144 
5.5.4.1 Practical aspects for testing bacteria ....................................... 144 
5.5.4.2 Practical aspects for testing fungi............................................ 145 
5.5.5 Testing conditions for specific states .................................................. 146 
5.5.5.1 Wet-state preservation and curative treatments ....................... 146 
5.5.5.2 Protection of solid material: PT 7, 9, 10 ................................... 146 
5.5.6 PT6 Preservatives for products during storage ..................................... 149 
5.5.7 PT 7 Film preservatives and PT 9 Fibre, rubber and polymerised materials 

preservatives ................................................................................ 150 
5.5.7.1 Simulation Tests (Tier 1 testing) ............................................. 151 
5.5.7.2 Tests based on artificial growth media (Tier 1 testing) ............... 152 
5.5.7.3 Tier 2 Testing ....................................................................... 153 
5.5.7.4 Tier 3 Testing ....................................................................... 154 
5.5.7.5 Prevention of Odour by odour-producing micro-organisms ......... 154 
5.5.8 PT8 Wood preservatives ................................................................... 158 
5.5.8.1 Label claims ......................................................................... 158 
5.5.8.1.1 User Category (Code for Product A.xx) ....................... 159 
5.5.8.1.2 Wood Category (Code for product B.xx) ...................... 160 
5.5.8.1.3 Wood Product (Code for product C.xx) ........................ 160 
5.5.8.1.4 Application aim and field of use.................................. 160 
5.5.8.1.5 Method of application and application rate (Code for product 
F.xx): ....................................................................... 162 

 
 
 
 
 
   
 
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5.5.8.1.6 Target organisms (Code for product G.xx) ................... 163 
5.5.8.1.7 Examples of a claimed matrix .................................... 165 
5.5.8.2 Available data ...................................................................... 166 
5.5.8.2.1 Standard test mthods ............................................... 166 
5.5.8.2.2 Preventive treatments .............................................. 170 
5.5.8.2.3 Curative treatment ................................................... 175 
5.5.8.2.4 Resistance ............................................................... 176 
5.5.8.3 Biological re-testing after changing the product formulation ....... 176 
5.5.9 PT9 Fibre, rubber and polymerised materials preservatives ................... 177 
5.5.10 PT10 Construction material preservatives ......................................... 177 
5.5.11 PT11 Preservatives for liquid-cooling and processing systems .............. 177 
5.5.12 PT12 Slimicides ............................................................................. 177 
5.5.13 PT13 Working or cutting fluid preservatives ...................................... 177 
5.6 PEST CONTROL (MAIN GROUP 3) ............................................................... 178 
5.6.1 General .......................................................................................... 178 
5.6.2 PT14 Rodenticides ........................................................................... 178 
5.6.2.1 Introduction ......................................................................... 178 
5.6.2.1.1 Aim ........................................................................ 179 
5.6.2.1.2 Global structure of the assessment ............................. 179 
5.6.2.2 Dossier Requirements ........................................................... 179 
5.6.2.2.1 Test animals ............................................................ 182 
5.6.2.2.2 Laboratory studies for bait products ........................... 182 
5.6.2.2.3  Laboratory  studies  related  to  contact  rodenticides  and 

gassing agents ........................................................... 183 

5.6.2.2.4  Laboratory  studies  related  to  specific  efficacy  claims 
regarding  suitability  of  bait  products  for  use  in  damp 
conditions ................................................................. 185 

5.6.2.2.5  Studies  related  to  specific  efficacy  claims  regarding  to 

the shelf life of bait products ....................................... 186 
5.6.2.2.6 Field trial and semi field trial ..................................... 186 
5.6.2.2.7 Waivers .................................................................. 188 
5.6.2.2.8 Biocidal Product Families (BPF) .................................. 188 
5.6.2.3 Methodology of assessment ................................................... 189 
5.6.2.4 Assessment of authorisation................................................... 189 
5.6.2.4.1 Norms and criteria ................................................... 189 
5.6.2.5 References for PT14 .............................................................. 190 

5.6.3  PT15  Avicides,  PT16  Molluscicides,  vermicides  and  products  to  control 

other invertebrates & PT17 Piscicides ............................................... 190 
5.6.4 PT18 Insecticides, acaricides and products to control other arthropods ... 190 
5.6.4.1 Introduction ......................................................................... 190 
5.6.4.1.1 Aim ........................................................................ 190 
5.6.4.1.2 Global structure of the assessment ............................. 191 
5.6.4.1.3 Dossier requirements................................................ 191 
5.6.4.1.4 Methodology of assessment ....................................... 196 
5.6.4.1.5 Assessment of authorisation ...................................... 197 
5.6.4.2 General Claims: Crawling Insects, Flying Insects, Acaricide ........ 198 
5.6.4.2.1 Introduction ............................................................ 198 
5.6.4.2.2 Dossier requirements................................................ 198 
5.6.4.2.3 Assessment of authorisation ...................................... 199 
5.6.4.3 Cockroaches ........................................................................ 199 
5.6.4.3.1 Introduction ............................................................ 199 

 
   
 
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5.6.4.3.2 Dossier requirements................................................ 200 
5.6.4.3.3 Assessment of authorisation ...................................... 203 
5.6.4.4 Ants .................................................................................... 204 
5.6.4.4.1 Introduction ............................................................ 204 
5.6.4.4.2 Dossier requirements................................................ 204 
5.6.4.4.3 Assessment of authorisation ...................................... 207 
5.6.4.5 Termites .............................................................................. 208 
5.6.4.5.1 Introduction ............................................................ 208 
5.6.4.5.2 Dossier requirements................................................ 210 
5.6.4.5.3 Assessment of authorisation ...................................... 212 
5.6.4.6 Bed Bugs ............................................................................. 213 
5.6.4.6.1 Introduction ............................................................ 213 
5.6.4.6.2 Dossier requirements................................................ 213 
5.6.4.6.3 Assessment of authorisation ...................................... 215 
5.6.4.7 Ticks ................................................................................... 215 
5.6.4.7.1 Introduction ............................................................ 215 
5.6.4.7.2 Dossier requirements................................................ 216 
5.6.4.7.3 Assessment of authorisation ...................................... 218 
5.6.4.8 Mites ................................................................................... 218 
5.6.4.8.1 Introduction ............................................................ 218 
5.6.4.8.2 Dossier requirements................................................ 219 
5.6.4.8.3 Assessment of authorisation ...................................... 220 
5.6.4.9 Fleas ................................................................................... 220 
5.6.4.9.1 Introduction ............................................................ 220 
5.6.4.9.2 Dossier requirements................................................ 221 
5.6.4.9.3 Assessment of authorisation ...................................... 222 
5.6.4.10 Litter Beetles ...................................................................... 222 
5.6.4.10.1 Introduction........................................................... 222 
5.6.4.10.2 Dossier requirements .............................................. 223 
5.6.4.10.3 Assessment of authorisation .................................... 223 

5.6.4.11  Textile-attacking  Insects  (including  fur  and  fabric  attaching 

insects) ................................................................................ 224 
5.6.4.11.1 Introduction........................................................... 224 
5.6.4.11.2 Dossier requirements .............................................. 224 
5.6.4.11.3 Assessment of authorisation .................................... 225 
5.6.4.12 Stored Goods-attacking Insects and Mites .............................. 225 
5.6.4.12.1 Introduction........................................................... 225 
5.6.4.12.2 Dossier requirements .............................................. 226 
5.6.4.12.3 Assessment of authorisation .................................... 227 
5.6.4.13 Flies .................................................................................. 227 
5.6.4.13.1 Introduction........................................................... 227 
5.6.4.13.2 Dossier requirements .............................................. 228 
5.6.4.13.3 Assessment of authorisation .................................... 231 
5.6.4.14 Mosquitoes ......................................................................... 231 
5.6.4.14.1 Introduction........................................................... 231 
5.6.4.14.2 Dossier requirements .............................................. 232 
5.6.4.14.3 Assessment of authorisation .................................... 235 
5.6.4.15 Wasps ............................................................................... 236 
5.6.4.15.1 Introduction........................................................... 236 
5.6.4.15.2 Dossier requirements .............................................. 236 
5.6.4.15.3 Assessment of authorisation .................................... 237 

 
 
 
 
 
   
 
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5.6.5 PT19 Repellents and attractants ........................................................ 238 
5.6.5.1 Introduction ......................................................................... 238 
5.6.5.1.1 Aim ........................................................................ 238 
5.6.5.1.2 Global structure of the assessment ............................. 238 
5.6.5.1.3 Dossier requirements................................................ 239 
5.6.5.1.4 Methodology of assessment ....................................... 244 
5.6.5.1.5 Definitions to determine Complete Protection Time (CPT) 247 
5.6.5.1.6 Assessment of authorisation ...................................... 248 
5.6.5.2 Ants .................................................................................... 249 
5.6.5.2.1 Introduction ............................................................ 249 
5.6.5.2.2 Dossier requirements................................................ 249 
5.6.5.2.3 Assessment of authorisation ...................................... 255 
5.6.5.3 Bed bugs ............................................................................. 255 
5.6.5.3.1 Introduction ............................................................ 255 
5.6.5.3.2 Dossier requirements................................................ 256 
5.6.5.3.3 Assessment of authorisation ...................................... 261 
5.6.5.4 Biting midges (Culicoides, veterinary) ..................................... 262 
5.6.5.4.1 Introduction ............................................................ 262 
5.6.5.4.2 Dossier requirements................................................ 263 
5.6.5.4.3 Assessment of authorization ...................................... 266 
5.6.5.5 Cockroaches ........................................................................ 266 
5.6.5.5.1 Introduction ............................................................ 266 
5.6.5.5.2 Dossier requirements................................................ 267 
5.6.5.5.3 Assessment of authorisation ...................................... 271 
5.6.5.6 Fleas ................................................................................... 271 
5.6.5.6.1 Introduction ............................................................ 271 
5.6.5.6.2 Dossier requirements................................................ 272 
5.6.5.6.3 Assessment of authorisation ...................................... 279 
5.6.5.7 Flies on grazing cattle, horses and other livestock ..................... 280 
5.6.5.7.1 Introduction ............................................................ 280 
5.6.5.7.2 Dossier requirements................................................ 281 
5.6.5.7.3 Assessment of authorization ...................................... 285 
5.6.5.8 Fruit Flies and Scuttle Flies .................................................... 285 
5.6.5.8.1 Introduction ............................................................ 285 
5.6.5.8.2 Dossier requirements................................................ 286 
5.6.5.8.3 Assessment of authorisation ...................................... 291 
5.6.5.9 Mosquitoes .......................................................................... 292 
5.6.5.9.1 Introduction ............................................................ 292 
5.6.5.9.2 Dossier requirements................................................ 292 
5.6.5.9.3 Assessment of authorisation ...................................... 305 
5.6.5.10 Stable flies indoor ............................................................... 306 
5.6.5.10.1 Introduction........................................................... 306 
5.6.5.10.2 Dossier requirements .............................................. 307 
5.6.5.10.3 Assessment of authorisation .................................... 309 
5.6.5.11 Stored goods-attacking insects and mites .............................. 310 
5.6.5.11.1 Introduction........................................................... 310 
5.6.5.11.2 Dossier requirements .............................................. 312 
5.6.5.11.3 Assessment of authorisation .................................... 316 

5.6.5.12  Textile-attacking  insects  (including  fur  and  fabric  attacking 

insects) ................................................................................ 317 
5.6.5.12.1 Introduction........................................................... 317 

 
   
 
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5.6.5.12.2 Dossier requirements .............................................. 317 
5.6.5.12.3 Assessment of authorisation .................................... 322 
5.6.5.13 Ticks ................................................................................. 323 
5.6.5.13.1 Introduction........................................................... 323 
5.6.5.13.2 Dossier requirements .............................................. 324 
5.6.5.13.3 Assessment of authorisation .................................... 333 
5.6.5.14 Wasps ............................................................................... 333 
5.6.5.14.1 Test species ........................................................... 333 
5.6.5.14.2 Requirements per type of claim and test methods ...... 333 
5.6.5.14.3 Assessment of authorisation .................................... 334 
5.6.6 PT20 Other vertebrates .................................................................... 334 
5.7 OTHER BIOCIDAL PRODUCTS (MAIN GROUP 4) ............................................ 334 
5.7.1 PT21 Antifouling products ................................................................. 334 
5.7.1.1 General Introduction ............................................................. 334 
5.7.1.1.1 Introduction ............................................................ 334 
5.7.1.1.2 Types of Coating ...................................................... 334 
5.7.1.1.3 Mode of Action ......................................................... 334 
5.7.1.1.4 Categorisation of antifouling products ......................... 335 
5.7.1.1.5 Spectrum of activity ................................................. 335 
5.7.1.1.6 Dossier requirements................................................ 335 
5.7.1.1.7 Label claims ............................................................ 335 
5.7.1.1.8 Efficacy tests ........................................................... 336 
5.7.1.1.9 Standard test methods ............................................. 338 
5.7.1.1.10 Resistance ............................................................. 339 
5.7.1.1.11  Reports  of  development  of  resistance  should  always 

be mentioned. Service life ........................................... 339 
5.7.1.2 Products intended for marine use ........................................... 339 
5.7.1.2.1 Introduction ............................................................ 339 
5.7.1.2.2 Dossier requirements................................................ 340 
5.7.1.2.3 Assessment of authorisation ...................................... 340 
5.7.1.3 Products for freshwater use ................................................... 341 
5.7.1.3.1 Introduction ............................................................ 341 
5.7.1.3.2 Dossier requirements................................................ 341 
5.7.1.3.3 Assessment of authorisation ...................................... 341 
5.7.1.4 Products for use in aquaculture .............................................. 342 
5.7.1.4.1 Introduction ............................................................ 342 
5.7.1.4.2 Dossier requirements................................................ 342 
5.7.1.4.3 Assessment of authorisation ...................................... 342 
5.7.2 PT22 Embalming and taxidermist fluids .............................................. 343 
5.7.2.1 General introduction ............................................................. 343 
5.7.2.2 Use of the products ............................................................... 343 
5.7.2.2.1 The issue of bodily decomposition .............................. 343 
5.7.2.2.2 Products for preserving human bodies and their uses ... 344 
5.7.2.3 Data required ....................................................................... 345 
5.7.2.3.1 Claims and labelling ................................................. 345 
5.7.2.3.2 Efficacy tests ........................................................... 345 
5.7.2.4 Assessing the application for authorisation ............................... 348 

APPENDIX 1. CLAIMS MATRICES ......................................................................... 349 

APPENDIX 2. STANDARDS AND TESTING METHODS FOR EFFICACY-TESTING OF 
DISINFECTANT BIOCIDAL PRODUCTS (PT 1-5) ...................................................... 350 

 
 
 
 
 
   
 
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APPENDIX 3. TABLE OF REFERENCE TEST ORGANISMS (PT 1-5).............................. 358 

APPENDIX  4.  OVERVIEW  OF  STANDARDS,  TEST  CONDITIONS  AND  PASS  CRITERIA 
(PT 1-5) ........................................................................................................... 360 

APPENDIX 5. EXAMPLES OF VIRUSES SORTED ACCORDING TO THEIR PRESENCE IN THE 
HUMAN BODY IN CASE OF VIRUS INFECTION ....................................................... 378 

APPENDIX 6. SELECTION OF RECOMMENDED TESTS FOR SOLID MATERIALS (EXCLUDING 
WOOD-PRESERVATIVES) .................................................................................... 380 

APPENDIX 7. SELECTION OF RECOMMENDED TESTS FOR LIQUID MATERIALS ........... 383 

APPENDIX 8. COMMONLY USED METHODS TO MEASURE THE EFFECTS OF 
PRESERVATIVE/CURATIVE ACTION IN LIQUID MATRICES ....................................... 384 

APPENDIX 9. COMMONLY USED METHODS TO MEASURE THE EFFECTS OF PROTECTING 
MATERIAL ......................................................................................................... 386 

APPENDIX 10. COMMONLY USED METHODS TO MEASURE ANTIMICROBIAL ACTIVITY  401 

APPENDIX 11. INFORMATION ON THE PRINCIPLE TARGET ORGANISMS FOR PT 8 AS 
OUTLINED IN THE DOCUMENT (5.5.8) ................................................................. 410 

APPENDIX 12. ANNEX A OF EN 599-1 ................................................................... 413 

APPENDIX 13. LABORATORY STUDIES FOR RODENTICIDES : BAIT CHOICE TEST ...... 419 

APPENDIX 14. FIELD TRIAL FOR RODENTICIDE BAITS ........................................... 421 

APPENDIX 15. LIST OF CURRENTLY AVAILABLE STANDARD TEST METHODS FOR 
RODENTICIDES ................................................................................................. 424 

APPENDIX 16. ADDITIONAL INFORMATION ON LABEL CLAIMS ................................ 427 

APPENDIX 17. SPECIES GRID .............................................................................. 434 

APPENDIX 18. LIST OF CURRENTLY AVAILABLE STANDARD TEST METHODS FOR PRODUCT 
TYPE 18 INSECTICIDES/ACARICIDES AND PRODUCT TYPE 19 REPELLENTS/ATTRACTANTS 
(AS FAR AS THEY CONCERN INSECTS AND OTHER ARTHROPODS) .......................... 441 

APPENDIX 19. EFFICACY GUIDELINE WITH COCKROACH; FIELD TRIAL .................... 465 

APPENDIX 20. CURRENT ANTIFOULING COATINGS ................................................ 467 

APPENDIX 21. PUBLISHED PAPER (CEPE ANTIFOULING WORKING GROUP) ............... 469 

APPENDIX 22. EXAMPLE OF HOW AN OVERALL FOULING ASSESSMENT MAY BE CARRIED OUT 
FOR PANEL TESTING IN MARINE WATERS ............................................................ 475 

APPENDIX 23. PT 22 ACTIVE SUBSTANCES IN THE REVIEW PROGRAMME ................. 477 

APPENDIX 24. ASSESSMENT GRID FOR TESTS ON HUMAN BODIES ......................... 478 

Figures 

Figure 1: Decision scheme to distinguish between claims for material protection and claims 
for protection of humans and animals .................................................................. 47 
Figure 2: The various phases of a cycle of disinfection of an automatic process ......... 73 
Figure 3: A Test for Antibacterial Activity in Wet Conditions .................................... 133 
Figure 4: Simulated Splash Model Non-Porous Materials ......................................... 135 
Figure 5: Simulated Splash Model Porous Materials ................................................ 136 

 
   
 
 
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Figure 6: Printing Model ..................................................................................... 137 
Figure 7: Decision scheme for the distinction between preservation/curative action 

and disinfection ................................................................................... 141 
Figure 8: Example of a Simulated Growth Test ...................................................... 152 
Figure 9: An Example of an Agar Plate Based Test ................................................. 153 
Figure 10: OECD/IBRG Tier 1 Textile Test ............................................................. 156 
Figure 11: Life cycle of subterranean termites ....................................................... 209 
Figure  12:  Example  for  the  experimental  set-up  of  the  laboratory  choice  test  for 
repellents against ants.  
Figure  13:  Example  for  the  experimental  set-up  (A:  before  and  B:  after  product 
application) of the simulated-use test for repellents against ants 
Figure  14:  Room  test  volunteer  wearing  a  light  beekeeper  suit,  gloves  and  white 
hospital booties and one arm is protected by a “tube” of flexible material 
Figure  15:  Upper  side  of  a  human  test  arm  in  a  simulated-use  test  to  show 
repellency against ticks 

252 

251 

297 

327 

Tables 

Table 1: Example ready-to-use disinfectants with/without pre-cleaning*. ................. 39 
Table 2: Example concentrated disinfectants ......................................................... 40 
Table 3: Example surface disinfectants ready-to-use: more PT’s .............................. 40 
Table 4: Example insecticide: take target organisms and application method into 

account. ............................................................................................... 41 
Table 5: Example disinfectant: take formulation into account. ................................. 42 
Table 6: Example anti-fouling product: Different ratios of two (or more) active 

substances............................................................................................ 43 

Table 7: Number of sampling points ..................................................................... 116 
Table 8: Number of sampling points ..................................................................... 125 
Table 9: Protection of Humans or Animals – Example Claims, Problems and Testing 

Approaches ........................................................................................... 137 
Table 10: Basic Requirements for a Valid Test Protection of Humans or Animals ........ 139 
Table 11: Examples ........................................................................................... 148 
Table 12: Basic Requirements for a Valid Test Protection ........................................ 154 
Table 13: Odour: Example Claims, Problems and Testing Approaches ...................... 157 
Table 14: Different categories and the related product codes .................................. 159 
Table 15: User categories ................................................................................... 159 
Table 16: Wood categories ................................................................................. 160 
Table 17: Wood product categories ...................................................................... 160 
Table 18: Application aim ................................................................................... 161 
Table 19: Different fields of uses ......................................................................... 162 
Table 20: Method of application ........................................................................... 162 
Table 21: Examples of target organisms for wood preservatives ......................... 164 
Table 22: Examples of claim matrix based on the application codes for product ......... 165 
Table 23: Preventive treatments: List of available standards and others methods 

used in wood preservation .................................................................... 168 

Table 24: Curative treatments: List of available standards used in wood curative treatments 
(based on EN 14128) ......................................................................................... 170 
Table 25: Toxicity ranking of known active substances used in anticoagulant 

rodenticides based on LD 50 (acute) data of brown rats and house mice 
compiled from CA-Reports, ranking from high (1) to lower toxicity (3) ....... 188 
Table 26: Target organisms versus test organisms ................................................ 205 

 
 
 
 
 
   
 
 
 
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Table 27: Overview guidelines on termites ............................................................ 212 
312 
Table 28: Examples of different pheromones that are used to attract food moths 
313 
Table 29: General claims based on target organisms 
Table 30: Overview of the required test species depending on the claim 
324 
Table 31: CEN European standards ...................................................................... 350 
Table 32: Other test methods and guidance documents ......................................... 354 
Table 33: Reference Test Organisms .................................................................... 358 
Table 34: Examples of viruses ............................................................................. 378 
Table 35: Selection of recommended tests for solid materials (excluding wood-
preservatives) ................................................................................................... 380 
Table 36: Selection of recommended tests for liquid materials ................................ 383 
Table 37: Commonly Used Methods to Measure the Effects of Preservative/Curative 
Action in Liquid Matrices ..................................................................................... 384 
Table 38: List of standards ................................................................................. 424 
Table 39: Components Making Up a Label Claim .................................................... 431 
Table 40: Example of linking label claims ............................................................. 433 
Table 41: PT 18 Crawling Insects ......................................................................... 434 
Table 42: PT 18 Flying Insects ............................................................................ 439 
Table 43: General .............................................................................................. 442 
Table 44: Crawling Insects: Cockroaches .............................................................. 444 
Table 45: Crawling Insects: Termites ................................................................... 445 
Table 46: Crawling Insects: Other Crawling Insects ............................................... 447 
Table 47: Flying Insects ..................................................................................... 448 
Table 48: Insecticides Against Textile and Stored Product Pests .............................. 450 
Table 49: Repellents & Attractants ....................................................................... 452 
Table 50: Current Antifouling Coatings ................................................................. 467 
Table 51: Example of categorisation of fouling coverage into ratings from 0 to 4 ....... 475 
Table 52: Example of weighting of ratings ............................................................ 475 
Table 53: Example of categorisation of overall efficacy ........................................... 476 
Table 54: PT 22 active substances in the review programme ................................... 477 

 
   
 
 
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NOTES to the reader:  

In this document text cited from the Biocidal Products Regulation (EU) No 528/2012 
is indicated in green boxes. 

•  This symbol highlights text to be noted. 

Section  5.6  and  sub-sections  for  PT10,  PT11,  PT12,  PT15,  PT16,  PT17  and  PT20: 
please refer to the General sections 1-3 of this guidance and the TNsG. 

 
 
 
 
 
   
 
 
 
 
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List of Abbreviations 

Abbreviation  

Explanation  

AACC 
International 

American Association of Cereal Chemists 
New name: Cereals & Grains Association 
https://www.cerealsgrains.org 

AATCC 

AFNOR 

AFPP 

AOAC 

AS 

ASTM 

ATCC 

BBA 

BP 

BPD 

BPR 

BSI 

CA 

CAR 

Cefic 

CEN 

American Association of Textile Chemists and Colorists 
https://www.aatcc.org 

French  Association  for  Standardisation  (Association  Française  de 
Normalisation) http://www.afnor.org  

Association Française de Protection des Plantes 
New name: Vegephyl - Association for plant health 
https://www.vegephyl.fr 

Association of Official Analytical Collaboration (AOAC) INTERNATIONAL 
http://www.aoac.org  

Active substance 

American Society for Testing Materials (ASTM) INTERNATIONAL 
http://www.astm.org  

American Type Culture Collection 
http://www.lgcstandards-atcc.org 

Federal  Research  Centre  for  Cultivated  Plants  (Julius  Kühn-Institut  - 
Bundesforschungsinstitut für Kulturpflanzen) 
https://www.julius-kuehn.de 

Biocidal product 

Biocidal Products Directive 98/8/EC 

Biocidal Products Regulation (EU) No 528/2012 

British Standard Institution (BS standards) 
https://www.bsigroup.com 

Competent Authority 

o  Evaluating CA (eCA) is the Competent Authority that evaluates 
the  application  for  an  active  substance  approval  or  an 
application for a Union authorisation.  

o  Receiving  CA  is  the  Competent  Authority  that  receives  an 

application for a National Authorisation. 

Competent Authority Report 

European Chemical Industry Council 
https://cefic.org/ 

European Committee for Standardisation 
http://www.cen.eu 

 
   
 
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Abbreviation  

Explanation  

Guidance on the BPR: Volume II Parts B+C  
Version 4.0 December 2021 

CEPE 

cfu 

CIP 

CSMA 

CT 

CTBA 

Ctgb 

CV 

DG SANCO 

DIN 

DVG 

ECDC 

EMA 

EN 

EPA 

EPPO 

ESL 

EU 

European  Council  of  the  Paint,  Printing  Ink,  and  Artist’s  Colours 
Industry 
https://www.cepe.org/ 

Colony forming unit 

Cleaning-in-Place 

Chemical Specialties Manufacturers Association 

Contact time 

Technical Center for Wood and Furniture  
New name: Technological Institute (L’institut technologique FCBA) 
https://www.fcba.fr 

Board  for  the  Authorisation  of  plant  protection  products  and  biocides 
(Netherlands) 
https://www.ctgb.nl 

Critical value  
Directorate-General Health and Consumer Protection 
New name: Directorate-General for Health and Food Safety (SANTE) 
German 
Normung) 
http://www.din.de/ 

for  Standardization  (Deutsches 

Institute 

Institut 

für 

German Veterinary Medical Society (Deutsche Veterinärmedizinischen 
Gesellschaft) 
http://www.dvg.net 

European Centre for Disease Prevention and Control 

European Medicines Agency 
https://www.ema.europa.eu/ 

European Standard 

United States Environmental Protection Agency 
https://www.epa.gov/ 

European and Mediterranean Plant Protection Organization 
https://www.eppo.int 

Estimated service life 

European Union + Norway, Iceland and Lichtenstein 

Please note the BPR applies to the European Economic Area (EEA) and 
thus all references to the EU in the text should be understood as EEA 
(EU + Norway, Iceland and Lichtenstein) 

FCBA 

Technological Institute (L’institut technologique FCBA) 

https://www.fcba.fr/ 

GLP 

Good laboratory practice 

 
 
 
 
 
   
 
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Abbreviation  

Explanation  

ISO 

KD 

KD50 

KT50 

LD50 

MS 

OCSPP 

OECD 

OPPTS 

prEN 

PAR 

PT 

SABS 

SANTE 

RIVM 

SPC 

TNsG 

TVC 

UC 

US EPA 

VAH 

VOC 

International Organization for Standardisation 
http://www.iso.org 

Knock down 

Knock down for 50% of the group of tested animals 

Knock down time for 50% of the group of tested animals  

Lethal dose for 50% of the group of tested animals 

Malaysian Standard 
https://ikm.org.my/sda/ 

Office of Chemical Safety and Pollution Prevention (EPA) 
https://www.epa.gov/aboutepa/about-office-chemical-safety-and-
pollution-prevention-ocspp 

Organisation for Economic Co-operation and Development 
http://www.oecd.org 
Office of Prevention, Pesticides and Toxic Substances 
New  name:  Office  of  Chemical  Safety  and  Pollution  Prevention 
(OCSPP, please see above) 

Draft European Standard 

Product Assessment Report 

Product type 

South African Bureau of Standards 
https://www.sabs.co.za/ 

Directorate-General for Health and Food Safety 

National Institute for Public Health and the Environment (Netherlands) 
https://www.rivm.nl/ 

Summary of Product Characteristics 

Technical Notes for Guidance 

Total viable count 

Use Class 

United States Environmental Protection Agency 
http://www.epa.gov/ 

Association for Applied Hygiene (Verbund für Angewandte Hygiene) 
http://www.vah-online.de/ 

Volatile organic compound 

 
   
 
 
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Glossary of Terms 

Standard term  

Explanation  

Activity against 
enveloped viruses 
(see also Virucidal 
activity and Limited 
spectrum virucidal 
activity) 

Algaecide 

A  claim  for  hygienic  hand  and  skin  disinfectants  with  activity 
against enveloped viruses only. 

A product or active substance used to control (inhibit the growth) 
or kill algae. 

Algaecidal activity   The  capability  of  a  product  or  active  substance  to  produce  a 
reduction  in  the  number  of  viable  algae  cells  under  defined 
conditions. 

Antimicrobial 
product 

Bactericide 

Bactericidal 
activity 

A  product  which  prevents  the  growth  of/reduces  the  number 
of/mitigates the growth of micro-organisms 

A  product  or  active  substance  which  irreversibly  inactivates 
vegetative bacteria under defined conditions 

The  capability  of  a  product  or  active  substance  to  produce  a 
reduction  in  the  number  of  viable  bacterial  cells  of  relevant  test-
organisms under defined conditions 

Bacteriostatic 
activity 

Capability of a product or active substance to inhibit the growth of 
bacteria under defined conditions 

Biocidal product/ 
Biocide 

BPR Article 3(1)(a): 
— any substance or mixture, in the form in which it is supplied to 
the  user,  consisting  of,  containing  or  generating  one  or  more 
active  substances,  with  the  intention  of  destroying,  deterring, 
rendering  harmless,  preventing  the  action  of,  or  otherwise 
exerting  a  controlling  effect  on,  any  harmful  organism  by  any 
means other than mere physical or mechanical action,  
—  any  substance  or  mixture,  generated  from  substances  or 
mixtures which do not themselves fall under the first indent, to be 
used  with  the  intention  of  destroying,  deterring,  rendering 
harmless,  preventing  the  action  of,  or  otherwise  exerting  a 
controlling  effect  on,  any  harmful  organism  by  any  means  other 
than mere physical or mechanical action.  
A  treated  article  that  has  a  primary  biocidal  function  shall  be 
considered a biocidal product. 

Biofilm 

An  accumulation  of  microbial  cells  immobilised  on  a  substratum 
and embedded in an organic polymer matrix of microbial origin 

Biostatic product 

A  product  which  inhibits  the  growth  of  micro-organisms  under 
defined conditions 

Curative effect on 
biofilm 

The biocide is added after the biofilm is formed and acts on biofilm 
stability,  facilitating  the  biocide  interaction  with  cells  –  it  may  or 
may not act as detergent and detach the biofilm from the surface 

 
 
 
 
 
   
 
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Standard term  

Explanation  

Disinfectant within 
PT 2, 3, 4 and 5 

A  disinfectant  is  a  product  that  reduces  the  number  of  micro-
organisms  in  or  on  an  inanimate  matrix-  achieved  by  the 
irreversible action of a product, to a level judged to be appropriate 
for a defined purpose  

Disinfection within 
PT 2, 3, 4 and 5 

disinfection  is  the  reduction  of  the  number  of  micro-organisms  in 
or on an inanimate matrix- achieved by the irreversible action of a 
product, to a level judged to be appropriate for a defined purpose 

Skin disinfection 
within PT1 

Skin  disinfection  is  the  reduction  of  the  number  of  micro-
organisms  on  skin,  achieved  by  the  irreversible  action  of  a 
product, to a level judged to be appropriate for a defined purpose 

Efficacy 

Flow condition (for 
biofilm) 

Fungicide 

The ability of a product or active substance to produce an effect as 
described in the label claims made for  it, when used under actual 
use conditions. 

Biofilm  is  formed  on  supports  of  different  nature  placed  along  a 
tube or a chamber where the medium (inoculated and/or fresh) is 
circulated in a closed (reservoir-pump-tubing) or  open (reservoir-
pump-tubing-outlet) system 

A  product  or  active  substance  which  irreversibly  inactivates  fungi 
(vegetative  mycelia,  budding  yeasts  and/or  their  spores)  under 
defined conditions 

Fungicidal Activity  The  capability  of  a  product  or  active  substance  to  produce  a 
reduction in the number of viable vegetative yeast cells and mould 
spores of relevant test organisms under defined conditions 

Fungistatic 
activity 

The  capability  of  a  product  or  active  substance  to  inhibit  the 
growth of fungi under defined conditions 

Hygienic hand 
disinfectants 

A hygienic hand disinfectant is a hygienic handrub disinfectant or a 
hygienic hand wash disinfectant 

Hygienic handrub 
disinfectant 

Hygienic 
handwash 
disinfectant 

Limited spectrum 
virucidal activity 
(see also Virucidal 
activity and Activity 
against enveloped 
viruses) 

Log reduction / 
log10 reduction / 
lg reduction 

product  used  for  post-contamination  treatment  that  involves 
rubbing  hands,  without  the  addition  of  water,  which  is  directed 
against  transiently  contaminating  micro-organisms  to  prevent 
their transmission regardless of the resident skin flora 

product  used  for  post-contamination  treatment  that  involves 
washing  hands  with  water,  which  is  directed  against  transiently 
contaminating  micro-organisms  to  prevent  their  transmission 
regardless of the resident skin flora 

Limited spectrum virucidal activity is a claim for hygienic hand and 
skin  disinfectants  using  Adenovirus  and  Murine  Norovirus  as  test 
organisms,  thus  including  activity  against  the  test  viruses  and  all 
enveloped viruses (see Appendix 5). 

Reduction  presented  in  a  logarithmic  scale.  Example  1:  when  a 
disinfection  reduces  108  bacteria  to  102  bacteria,  this  is  a  lg 
reduction  of  6.  Example  2:  when  a  disinfection  reduces  5.107 
fungal spores to 8.103 fungal spores this is a lg reduction of 3.79. 

 
   
 
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Standard term  

Explanation  

Microbes/micro-
organisms 

(including  vegetative  cells  bacterial  spores  and 
bacteria 
mycobacteria)  fungi  (including  yeasts,  moulds  and  fungal  spores) 
algae,  viruses  (including  bacteriophages),  protozoa  (including 
cysts and other permanent states), etc. 

Mycobactericide 

A  product  or  active  substance  which  irreversibly  inactivates 
mycobacteria under defined conditions 

Mycobactericidal 
activity 

The  capability  of  a  product  or  active  substance  to  produce  a 
reduction  in  the  number  of  viable  mycobacterial  cells  of  relevant 
test organisms under defined conditions 

Neutraliser 

Performance 
standard 

A  chemical  agent  or  formulation  which  suppresses  the  residual 
activity  of  an  disinfectant  within  a  test  but  does  not  inhibit  or 
inactivate micro-organisms 

Regulatory  or  scientific  standard  for  biocides  that  is  either 
quantitative  or  qualitative  (that  may  also  be  specified  in  the  test 
method)  by  which  a  decision  is  taken  on  the  acceptability  of  a 
claim. 

Preventive effect 
on biofilm 

The  biocide  is  present  before  the  biofilm  is  formed  and  may  act 
both on cell viability and/or on cell adhesion/biofilm maturation 

Product type (PT) 

Product types (PT) are defined in BPR annex V 

Sporicide 

A product or active substance which inactivates dormant bacterial 
spores under defined conditions 

Sporicidal activity  The  capability  of  a  product  or  active  substance  to  produce  a 
reduction in the number of viable bacterial spores of relevant test 
organisms under defined conditions 

Sporistatic activity  The  capability  of  a  product  to  inhibit  the  germination  of  dormant 

bacterial spores under defined conditions 

Static condition 
(for biofilm) 

Biofilm  is  formed  on  supports  such  as  microplates  without 
agitation  after  an  incubation  time  that  depends  on  the  micro-
organism considered 

Surgical hand 
disinfectants 

A surgical hand disinfectant is a surgical handrub disinfectant or a 
surgical hand wash disinfectant 

Surgical handrub 
disinfectant 

Surgical 
handwash 
disinfectant 

Treated article 

Product  used  for  preoperative  treatment  that  involves  rubbing 
hands, without the addition of water, which is directed against the 
flora  of  micro-organisms  on  hands  to  prevent  the  transmission  of 
micro-organisms into the surgical wound 

Product  used  for  preoperative  treatment  that  involves  washing 
hands  with  water,  which  is  directed  against  the  flora  of  micro-
organisms  on  hands  to  prevent  the  transmission  of  micro-
organisms into the surgical wound 

A  treated  article  is  any  substance,  mixture  or  article  which  has 
been  treated  with,  or  intentionally  incorporates,  one  or  more 
biocidal products 

Tuberculocide 

A  product  or  active  substance  which  irreversibly  inactivates 
Mycobacterium tuberculosis under defined conditions 

 
 
 
 
 
   
 
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Standard term  

Explanation  

Tuberculocidal 
activity 

The  capability  of  a  product  or  active  substance  to  irreversibly 
inactivate  Mycobacterium  tuberculosis,  demonstrated  by  the 
capability  to  produce  a  reduction  in  the  number  of  viable  cells  of 
the test organism Mycobacterium terrae under defined conditions 

Virucide 

A  product  or  active  substance  which  irreversibly  inactivates 
viruses under defined conditions 

Virucidal activity 
(see also Limited 
spectrum virucidal 
activity + Activity 
against enveloped 
viruses) 

The  capability  of  a  product  or  active  substance  to  produce  a 
reduction  in  the  number  of  infectious  virus  particles  of  relevant 
test organisms under defined conditions 
“Full  spectrum”  virucidal  activity  is  a  claim  for  biocidal  products 
using  relevant  test  organisms  and  thus  showing  activity  against 
the enveloped and non-enveloped viruses. 

Yeasticide 

A  product  or  active  substance  which  irreversibly  inactivates  yeast 
under defined conditions 

Yeasticidal activity  The  capability  of  a  product  or  active  substance  to  produce  a 
reduction in the number of viable vegetative yeast cells of relevant 
test organisms under defined conditions 

 
   
 
 
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1. General Introduction 

Evaluation and Assessment 

The process of evaluation of active substance applications is given in Article 8 (BPR) and the 
common  principles  for  the  evaluation  of  dossiers  for  biocidal  products  (including  the 
representative  biocidal  product  in  the  context  of  the  active  substance  approval)  is  given  in 
Annex VI (BPR). 

The  evaluating  or  receiving  CA  uses  the  data  submitted  in  support  of  an  application  for 
active  substance approval or authorisation of  a biocidal product to make a  risk assessment 
based on the proposed use of the (representative) biocidal product. The general principles of 
assessment are given in Annex VI (BPR) and the evaluation is carried out according to these 
general  principles.  The  evaluating  body  will  base  its  conclusions  on  the  outcome  of  the 
evaluation and decide whether or not the biocidal (representative) product complies with the 
criteria  for  authorisation  set  down  in  Article  19(1)(b)  and/or  whether  the  active  substance 
may be approved. 

Efficacy  data  are  a  fundamental  component  in  the  regulatory  management  and  decision 
making  process  for  biocidal  products.  Efficacy  data  are  required  to  establish  the  benefit 
arising  from  the  use  of  biocidal  products  and  must  be  balanced  against  the  risks  their  use 
poses to man and the environment. 

Authorisation of a biocidal product will only be granted according to Art. 19 (1) b of the BPR 
if that product is shown to be sufficiently effective. 

Even  for  the  requirement  to  limit  the  use  to  the  minimum  necessary  and  the  general 
requirement  of  sustainable  use  of  biocidal  products  (Art.  17  and  18  BPR),  it  is  crucial  that 
the biocide in questions delivers the expected effect. 

The information and data required relevant to the effectiveness of the active substance(s) to 
be employed in biocidal products are outlined in Annex II, BPR, title 1 No. 6 and 7 and title 2 
No 5 and 6. For biocidal products the data required are set out in Annex III, Title 1 No 6 and 
7, and title 2, No 6 and 7. 

These  general  sections  at  the  beginning  of  this  guidance,  (namely  sections  1,  2  and  3), 
provide  a  general  overview  for  the  efficacy  evaluation;  the  more  specific  requirements  for 
each Product Type (PT), which must be met and should be followed in the first instance, are 
described in the later sections. 

2. Claims 

2.1 Introduction 

The  evaluation  of  the  efficacy  of  a  biocidal  product  is  carried  out  in  order  to  determine 
whether the claims made for the activity of the active substance (within the product) or the 
product itself, are supported by suitable efficacy data. A claim is the precondition and base 
for efficacy testing. 

Claims should comprise of the description of the problem and the way it is suggested to be 
solved  by  the  biocidal  treatment.  Claims  include  information  given  in  an  active  substance 
dossier,  information  on  the  label  of  a  product,  information  provided  on  a  web-site  or  in 
product-associated leaflets. All claims should be consistent. 

 
 
 
 
 
   
 
 
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Claims  can  range  from  simple  to  complex,  depending  on  the  activity  and  benefits  the 
applicant wishes to claim as resulting from the use of the active substance/biocidal product. 
This should include as a minimum the following information: 

•  The purpose of the claim (e.g. prevent destruction of material by insect infestations, 

disinfect surface); 

•  The function of the product (e.g. insecticide, wood preservative, disinfectant, etc.); 

•  The (group of) target organisms which will be controlled; 

• 

In-use concentration; 

•  Use conditions and area of use; 

•  The effect which will result from using the product on the target organisms (e.g. kill, 

control, repel, prevent, etc.); 

•  Any products, organisms or objects to be protected. 

Some  examples  are  available  in  the  different  claim  matrices  and  PT  specific  guidance 
sections (see later sections).  

However  this  basic  information  can  be  supplemented  by  additional  claims  which  further 
describe the effects of the active substance/product where appropriate, such as: 

•  How fast the effect is produced; 

•  The duration of the effect (residuality) or lifespan; 

•  The  types  of  surface  on  which  the  product  can  be  used  (e.g.  hard  porous  and  non-

porous surfaces, softwood). 

For products used to treat articles, additional information should be provided: 

•  Durability of the effect in relation to the expected life-span of the treated article; 

•  Resilience  towards  ageing,  weathering  or  other  use  conditions  as  for  instance 

washing; 

•  Where  relevant,  leaching/migration  data  for  different  materials  or  different  use 

conditions.  

All  claims  made  should  be  supported  by  data  or  a  suitably  robust  scientifically  based 
reasoned case. 

2.2 Label claims and directions for use 

The  directions  for  use  and  the  claims  made  for  the  biocidal  product  are  included  in  a 
summary of biocidal product characteristics (SPC) in accordance with Article 22(2) (BPR). 

A label claim is information which is provided to the user which describes the biocidal effects 
that will result from using a biocidal product under its normal conditions of use (e.g. when it 
is  used  at  the  recommended  dose/application  rate,  by  the  recommended  application 
method(s) and in the appropriate areas, etc.). The product label can only include claims that 
are in line with the authorised uses, as given in the SPC. 

Label  claims  should  be  as  specific  as  possible,  or  if  more  general  claims  (such  as  “fast 
acting”)  are  made,  then  they  should  be  further  clarified  on  the  label  where  possible  (e.g. 
“fast  acting  –  acts  within  5  minutes”).  If  no  clarification  is  provided,  the  evaluating 
Competent Authority should ask the applicant to specify the claim. A judgement as to what a 
normal user would reasonably expect from the claim should be made. Evaluation should be 
made according to this claim and the directions for use should be taken into account. 

 
   
 
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An application  for  a product authorisation must include a  draft SPC and additionally  should 
include a copy of the draft product label containing the claims made for the product. 

Applications  for product families should include the entire range  of the claims proposed for 
the products within the family. 

3. General  considerations 
reporting of efficacy data 

3.1 Efficacy 

for 

the  development  and 

Efficacy  is  defined  as  the  ability  of  a  product  to  fulfil  the  claims  made  for  it  when  used 
according to the directions  for use  on the proposed product label (as given in the  SPC):  Is 
the product actually sufficiently effective against the claimed organisms under the conditions 
specified? The applicant must provide sufficient information to clearly specify the field of use 
of the product. In addition, studies must be provided to demonstrate that the product, when 
used  in  accordance  with  the  use  instructions  (concentration,  application  method,  etc.),  is 
sufficiently effective. 

3.1.1 Efficacy tests 

The  applicant  must  submit  studies  which  clearly  demonstrate  the  efficacy  of  the  active 
substance/product. 

We distinguish various types of studies: 

•  Screening tests  

•  Laboratory studies 

•  Simulation tests in laboratory 

•  Field tests 

Screening  tests  are  usually  not  related  to  practical/field  conditions  and  are  often  not 
implemented  with  the  complete  product  but  only  with  the  active  substance.  Such  tests  are 
therefore  primarily  useful  for  providing  supplementary  information,  for  example  to 
demonstrate that the concentration used is optimal. 

Laboratory  studies  are  performed  to  validate  the  efficacy  in  a  laboratory  according  to 
criteria  defined.  These  tests  permit  to  validate  for  example  a  level  of  mortality  during  a 
given time, a knock down (KD) effect and if need be the palatability of the product. 

Simulation  tests  are  more  linked  to  practical/field  conditions  and  can,  in  some  cases,  be 
sufficient  for  demonstrating  the  efficacy.  Simulation  tests  can  include  factors  like  ageing, 
weathering,  UV,  washing,  etc.  Example:  For  disinfecting  products  aimed  at  controlling 
bacteria on hard surfaces, it is sufficient to carry out a suspension test and a surface test in 
accordance with the relevant EN standards. 

Field  tests  provide  a  good  indication  of  how  the  product  works  in  practice/under  field 
conditions, to evaluate how the efficacy can be affected by a variety of factors (the weather, 
population  density,  natural  fluctuation  of  the  population  over  time  etc.).  The  experimental 
setup is important in these tests. The results of the tests should be compared to the results 
achieved  with  a  control  object  which  has  not  been  treated  or  with  the  situation  prior  to 
treatment:  however,  in  some  cases  it  is  not  possible  to  include  a  control  sample  in  field 
tests. 

 
 
 
 
 
   
 
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Screening  tests,  laboratory  studies  and  simulation  tests  must  always  include  an  untreated 
control  without  active  substance  (i.e.  a  negative  control);  it  is  preferred  that  this  is  the 
formulated product without active substance. However, providing it can be justified, this can 
be, a control with only the solvent, e.g. water. There are few exceptions to this rule, such as 
the EN disinfection test, and all exceptions should be justified by the methodology. 

Tests should preferably be carried out in accordance with standard protocols, e.g. CEN, ISO, 
OECD, ASTM, etc. If standard protocols are not available or are not suitable for the field of 
use  concerned,  other  methods  may  also  be  used  on  condition  that  the  studies  concerned 
have a sound scientific basis.  Preferably, available standard methods should be modified to 
meet  the  actual  application  in  such  cases.  Ideally,  tests  are  carried  out  in  accordance  with 
Good Laboratory Practice (GLP) or similar quality assurance systems (ISO), although this is 
not mandatory for efficacy tests. 

3.1.2 Test report 

Some  standard tests (e.g. EN tests) contain  examples  of appropriate reports,  which should 
be  used  as  a  template.  In  all  other  cases  the  test  report  must  contain  the  following 
elements: 

• 

introduction 

•  materials  and  methods  (e.g.  tested  product  composition,  conditions  of  the  test 

temperature, humidity,) 

tested organisms 

results and raw data 

conclusion/discussion based on criteria defined in guidance 

• 

• 

• 

The introduction must indicate the goal of the test. When a standard test is used the name 
and/or  number  of  the  test  should  be  stated.  The  section  on  materials  and  methods  must 
provide a complete description of the test method. If an internationally recognised standard 
method  is  used,  it  is  sufficient  to  provide  a  brief  description  of  the  test.  The  product  used 
and the concentration of the active substance must be specified. If the name of the product 
tested  is  not  the  same  as  the  product  for  which  the  application  is  being  submitted  (e.g.  a 
name  used  outside  the  EU  or  an  internal  company  code  for  the  product),  the  complete 
composition  of  the  product  tested  must  be  provided  in  a  separate  document.  The  test 
organisms used must correspond to the organisms against which the product is intended to 
be used, or they must be adequate representatives. For example, if a product is intended for 
use against bacteria in hospitals, it is not possible to test the product on all possible species 
of  bacteria.  Instead,  four  standard  species  of  bacteria  are  usually  tested.  The  conditions 
under which the negative control tests were carried out must also be described (e.g. treated 
with  product  not  containing  the  active  substance,  not  treated,  or  treated  with  water  for 
example). 

The  materials  and  methods  should  be  described  well.  In  case  of  standard  test  protocols  all 
the deviations should be indicated and justified. 

The  section  on  the  results  of  the  test  must  provide  quantitative  data.  It  is  not  sufficient  to 
present only tables or figures in which the results have been processed. The raw data must 
also  be  included.  In  case  of  repetitions  performed  in  the  test,  the  results  should  also  be 
subjected to a  statistical analysis, when appropriate. At the end of the report, a conclusion 
must be presented. Sometimes, it is necessary to discuss and/or present further arguments 
for  the  conclusion.  For  field  tests  in  particular,  the  results  obtained  in  repeated  tests  may 

 
   
 
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differ. If an  explanation is provided for  such differences in results, a test may possibly still 
be approved. 

Example:  In  test  1,  the  product  was  “washed  away  by  rainfall”  and  was  therefore  not 
effective,  but  tests  2  and  3  do  demonstrate  the  efficacy.  In  such  case  the  tests  can  be 
accepted and a remark will be made on  the SPC that the product should not be used when 
rain is expected within x hours, because this will influence the efficacy negatively. 

When applying for authorisation all the efficacy tests should be summarised in the PAR. The 
PAR format includes a table. This table should be filled out in a way that it gives an overview 
of  all  the  efficacy  results.  When  the  test  is  not  a  standard  test  a  short  description  of  the 
method  should  be  included.  The  test  column  “test  system/concentration  applied/  exposure 
time”  should  include  all  the  relevant  information  on  the  test,  the  test  parameter  (e.g. 
contact time, temperature,  replicates) in way  that it can be compared to the intended use. 
The  results  should  be  specified  (e.g.  x%  mortality,  log  reduction  >x)  and  not  just  “test 
passed”. In some cases it might be easier to summarise the results in the text instead of the 
table (e.g. field trials). 

Below  the  table  the  tests  should  be  discussed  and  an  explanation  should  be  given  on  how 
the  test  results  demonstrated  the  efficacy  of  the  product  for  the  different  uses  under  use 
conditions. 

3.2  Resistance 

The  topic  of  resistance  is  discussed  in  the  general  part  of  the  TNsG  on  Product  Evaluation 
(Section  6).  Information  on  resistance  should  be  given  for  active  substances  and  biocidal 
products.  Additionally,  in  support  of  the  review  for  each  active  substance,  information  on 
resistance is given in the Competent Authority Report (CAR) of this active substance. 

Resistance  will be assessed on the basis of  expert judgement. This section  of the  guidance 
will be updated in the future in the light of experience gained in evaluation of resistance. 

4. Active substance approval 

4.1 Introduction 

According  to  Article  4  of  the  BPR,  an  active  substance  must  be  approved  if  at  least  one 
biocidal product containing that active substance may be  expected to  meet the  criteria laid 
down in point (b) of article 19(1), and more particularly for the context of this guidance the 
paragraph (i), which says “the biocidal product is sufficiently effective”. 

During  the  review  of  an  active  substance  at  the  active  substance  approval  stage,  both  the 
efficacy of the active substance and of the representative biocidal product are assessed in a 
relevant matrix. At this approval stage, it is the activity of the active substance which must 
be demonstrated, both in its own right and when formulated into a biocidal product. 

Although  a  biocidal  product  containing  the  active  substance  is  evaluated  at  the  active 
substance  approval  stage,  this  part  of  the  BPR  process  is  concerned  primarily  with  the 
efficacy of the active substance itself. The purpose of this section is to provide guidance for 
applicants and competent authorities on the principles for evaluation of efficacy at the active 
substance  approval  stage,  and  to  help  determine  whether  the  information  provided  in  an 
application for approval of an active substance is sufficient for inclusion of the substance in 
the  Union  list.  For  guidance  on  data  requirement  see  Volume  II  Part  A  of  ECHA’s  guidance 
under the BPR. 

 
 
 
 
 
   
 
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4.2 General principles 

4.2.1 Intended use 

When making an application for approval of an active substance, the applicant must clearly 
describe the uses for which the active substance is intended. This information is required to 
allow  a  proper  evaluation  of  the  efficacy  to  be  carried  out,  and  must  include,  for  every 
product type separately: 

•  The purpose of the claim (e.g. prevent destruction of material by insect infestations, 

decrease risk of infection by bacterial contamination); 

•  The  function  of  the  active  substance  (e.g.  bactericide,  fungicide,  rodenticide, 

insecticide);  

•  The (group of) target organism(s) to be controlled; 

•  The effects on representative target organism(s) (e.g. attracting, killing, inhibiting); 

•  Any products, organisms or objects to be protected. 

•  The  likely  concentration  at  which  the  active  substance  will  be  used  in  products  and, 
where  appropriate, 
likely  concentration  should  be 
demonstrated  to  be  effective  according  to  the  requirements  described  in  section 
4.2.2.1. 

in  treated  articles.  This 

In  the  application,  the  applicant  may  choose  to  provide  information  on  all  of  the  intended 
target organisms at the active substance approval stage, or a representative selection. 

However,  in  order  for  approval  of  the  active  substance  to  be  granted,  efficacy  must  be 
demonstrated  for  at  least  one  main  target  organism  (or  group  of  target  organisms  e.g. 
bacteria).  Use  against  additional  target  organisms  may  be  applied  for  at  the  product 
authorisation stage. 

For  active  substances  used  in  treated  articles,  see  section  4.5  and  sub-sections  4.5.2  and 
4.5.3. 

4.2.2 Evaluation of efficacy 

Efficacy of an active substance has to be demonstrated both in part A of the CAR (related to 
the  intrinsic  efficacy  of  the  active  substance)  and  in  part  B  (where  the  active  substance  is 
incorporated in a formulated product). Evaluation of each part is described below. 

4.2.2.1 Active substance efficacy (part A): 

As  the  testing  of  an  active  substance  is  normally  carried  out  using  the  technical  active 
substance, or a simple dilution of the active substance in water or an appropriate matrix (so 
that  the  testing  is  carried  out  in  the  absence  of  other  substances  which  may  affect  the 
efficacy), an extensive data package and evaluation is not required at this stage. 

However,  efficacy  studies  should  be  submitted  on  the  active  substance,  and  these  data 
should  be  capable  of  demonstrating  the  innate  activity  of  the  active  substance  against 
representatives of the  proposed target organisms at the concentration relevant for the risk 
assessment. For that purpose, innate activity of an active substance could be defined as the 
capacity  of  an  active  substance  to  provide  a  sufficient  effect  on  one  or  several  relevant 
target organisms, for the use considered. 

The following minimum requirements should be fulfilled to demonstrate innate activity: 

 
   
 
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•  For  main  group  1  (disinfectants:  PT1,  2,  3,  4  and  5),  innate  activity  is  at  least  a 
“cidal”  activity  demonstrated  in  a  suspension  test  and  has  to  be  demonstrated 
against  one  or  more  representative  target  organism(s)  for  the  activity  claimed  (e.g. 
bactericide,  yeasticide),  preferably  according  to  the  CEN  norms  (phase  1  tests  and 
phase  2  step  1  tests).  Test  organism(s)  should  be  that  or  those  specified  in  the 
respective  norm.  Phase  1  tests  are  sufficient  for  the  active  substance  if  a  phase  2 
step  1  test  is  available  for  the  representative  product.  When  only  specific  biostatic 
activity (e.g. bacteriostatic, fungistatic) is claimed, an appropriate method should be 
used. 

•  For main group 2 (preservatives: PT6, 7, 8, 9, 10, 11, 12 and 13), innate activity is 
generally  a  static  activity  demonstrated  in  challenge  tests  on  several  and  relevant 
target  organisms,  in  the  relevant  matrix.  However,  if  curative  effects  are  claimed, 
cidal activity is requested. To demonstrate efficacy against one target organism only 
could also be acceptable in the case of a strictly defined use relevant for the PT ( e.g. 
the control of Legionella in cooling water in PT11). For PT8, CEN norms are available 
to support efficacy testing and give indications on representative target organisms to 
be tested. Growth in the untreated control is essential to show the validity of the test. 
If the claim is only for a curative effect, it is sufficient to show that the decline in the 
microbial  population  in  the  treated  samples  is  statistically  significantly  more  than  in 
the untreated control samples. 

•  For main group 3 (pest control: PT14, 15, 16, 17, 18, 19 and 20), innate activity can 
be  demonstrated  for  one  target  organism  only  (for  instance,  control  of  mice  or 
control of bedbugs). 

•  For  main  group  4  (other  biocidal  products:  PT21  and  PT22),  innate  activity  is 
generally supported on a group of organisms (algae, animals, bacteria) and examples 
of  appropriate  target  organisms  are  available  in  the  Efficacy  guidance  for  PT21  and 
PT22. 

When minimum requirements are not met this should be justified. 

Generally,  efficacy  data  are  generated  from  laboratory  tests,  performed  by  the  applicant. 
Nevertheless  efficacy  data  from  literature  could  also  be  acceptable  if  the  application  rate, 
target organisms, area of use and the identity of the active substance is described and are 
relevant.  If  cited  literature  is  used  to  support  a  preserving  effect  it  must  also  show  that 
untreated  test  specimens  supported  growth.  When  curative  effects  are  claimed  the  cited 
literature  must  demonstrate  the  efficacy  of  the  active  substance  according  to  the 
requirements per PT. The use of cited literature should be agreed between the applicant and 
the evaluation CA (eCA) on a case by case basis. 

The level of efficacy demonstrated at this stage of the process need not be high, as an active 
substance  in  a  simple  solution  may  not  be  as  effective  as  when  it  is  used  in  a  fully 
formulated  product.  For  that  reason  an  active  substance  should  still  be  considered  suitable 
for approval if the levels of efficacy demonstrated fulfil the minimum requirements above. In 
the case where the levels of efficacy of the active substance alone are lower than expected, 
efficacy  tests  performed  with  the  representative  product  has  to  show  a  sufficient/basic 
efficacy, according to the requirements above. If both are insufficient, approval for the Union 
list should not be proposed. 

If  no  efficacy  tests  with  the  active  substance  itself  are  available,  but  only  tests  with  a 
formulation, a justification has to be given by the applicant regarding the possible influence 
of co-formulants on the efficacy. If the co-formulants used potentially have biocidal activity, 

 
 
 
 
 
   
 
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it is essential to demonstrate that the efficacy is due to the active substance and not to the 
co-formulants,  e.g.  a  control  should  be  performed  with  all  co-formulants  but  without  the 
active substance. 

4.2.2.2 Product efficacy (part B): 

Although  approval  for  the  Union  list  is  primarily  concerned  with  the  active  substance, 
efficacy  data  is  also  required  for  a  representative  product.  Ideally  efficacy  data  on  an 
existing  biocidal  product  should  be  submitted.  If  this  is  not  possible  data  on  a  dummy 
product could be acceptable in order to demonstrate that the active substance is capable of 
producing  an  effect  on  the  target  organism  and  in  a  relevant  matrix  according  to  the 
proposed use, when included in a formulated product. 

However, a detailed evaluation of the effectiveness of the product (including an evaluation of 
the  proposed  label  claims)  is  not  in  all  cases  required  at  the  active  substance  approval 
stage. This may for example be the case where no marketed product is available. 

Nevertheless, the level of efficacy (e.g. the kind of activity “biocidal” or “biostatic”) have to 
be  consistent  with  the  uses  claimed  and  fulfil  the  minimum  requirements  mentioned  in  the 
active substance part (part A). 

4.2.3 Overall evaluation for active substance approval 

It  is  concluded  that  efficacy  data  are  required  on  the  active  substance,  to  demonstrate  on 
the  one  hand  the  innate  activity  of  the  substance  (either  the  technical  grade  active 
substance  or  a  dilution  in  water  or  a  solvent)  and  on  the  other  hand  the  efficacy  of  the 
representative  product  against  one  or  more  of  the  proposed  target  organisms.  Efficacy 
should be demonstrated in accordance with the use(s) considered in the risk assessment. If 
for  some  justified  reasons,  the  results  of  the  biocidal  product  do  not  completely  fulfil  the 
requirements  described  above,  this  could  still  be  acceptable  as  long  as  the  results  of  the 
active substance are sufficient to demonstrate efficacy. The other way around, if the results 
of the active substance do not fulfil the requirements described above acceptable data of the 
biocidal  product  may  be  sufficient  as  long  as  it  can  be  excluded  that  the  co-formulants 
contribute to the efficacy of the product. 

Where  the  levels  of  efficacy  demonstrated  are  low  enough  to  raise  concerns  by  the 
evaluating Member State, the applicant should be asked to justify why the result should still 
be  considered  acceptable.  Two  specific  reasons  are  discussed  below:  the  use  of  ‘dummy 
products’ and the  case  of active  substances not used alone but always in combination with 
other active substances. 

4.2.4 Link to risk assessment 

There is an essential link between efficacy testing and the risk assessment for human health 
and the environment at the active substance approval stage: 

•  Efficacy  has  to  be  proven  for  active  substance  concentrations  used  in  the  risk 

assessment 

•  Efficacy has to be sufficient for the use assessed in the risk assessment. 

The  information  on  efficacy  is  relevant  in  assessing  the  dose  recommended  for  the  use(s) 
applied  for.  The  dose  (or  the  "likely  concentration(s)  at  which  the  active  substance  will  be 
used" as stated in Annex II 6.4 of the BPR) is the starting point in the exposure assessment 
for human health and the environment. 

 
   
 
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4.3 Active substances which are not intended to be used in isolation 

This section is developed to deal with active  substances  which are not intended to be used 
as the sole active substance in a product. 

At the active substance approval stage, the following should be demonstrated: 

• 

in  part  A  (dedicated  to  the  active  substance),  the  innate  activity  of  the  active 
substance should be demonstrated against target organism(s) relevant for the field of 
use envisaged. 

The evaluation should demonstrate that the active substance is capable of producing an 
effect on its own or when formulated into a very simple product. Due to the absence of 
the  other  active  substance(s),  the  formulation  may  have  only  a  limited,  rather  than 
broad based, spectrum of activity, or a lower level of efficacy. 

Evaluation of the data will be done on a case by case basis. 

Some examples where limited efficacy could be acceptable: 

• 

• 

• 

• 

for  wood  preservatives  with  fungicidal  activity  where  different  fungicides  are  active 
against  different  groups  of  target  fungi  and  therefore  two  or  more  fungicides  would 
be included in a product to produce the full spectrum of antifungal activity; 

for insecticides that are used in combination with other active substances to improve 
the insecticidal performance of the latter as they exert a synergistic effect; 

for  insecticides  used  in  combination  with  a  co-formulants  (e.g.  booster)  that  is  not 
itself an active substance; 

the active substance is used in combination with another active substance. 

However,  an  appropriate  argumentation  is  always  required  in  order  to  justify  situations 
with a more restricted level  of efficacy.  The minimum requirements in section 4.2 have 
always to be fulfilled. 

• 

in part B (dedicated to the accompanying/representative product), the efficacy of a 
product where the active substance is  formulated in combination with  other (active) 
substances should be demonstrated against target organism(s) relevant for the field 
of  use  envisaged.  Relevant  efficacy  tests  should  be  used  and  structured  to  allow 
evaluation  of  the  contribution  of  the  active  substance  to  the  overall  efficacy.  This  is 
particularly important if efficacy data have not been submitted in part A. 

Efficacy data packages for formulations containing two or more active substances are not 
fully  suitable  for  determining  the  activity  contribution  from  the  active  substance  under 
evaluation.  For  that  reason  great  attention  should  be  paid  to  justify  the  contribution  of 
the  active  substance  under  evaluation  to  the  total  efficacy  of  the  product.  Information 
about the mode of action/function of the other active substances present in the product 
is also requested. 

The  submitted  data  should  allow  the  definition  of  an  effective  concentration  (i.e.  the 
concentration of active substance at the efficient application rate of the product) that can 
be  used  for  the  risk  assessment  (specified  per  use).  If  in  part  B  a  formulation  is 
introduced with additional co-active substances, this formulation will only be considered 
for efficacy testing and for setting a likely in-use  concentration of the active  substance, 
not used in isolation. 

 
 
 
 
 
   
 
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A  statement  should  be  added  in  the  BPC  opinion  in  order  to  stress  that  the  active 
substance  is  intended  to  be  used  in  combination  with  other  active  substances  or 
synergists. 

4.4 “Dummy products” 

A “dummy product” is a product that is not fully formulated. It is not intended to be placed 
on the market.  

In order to satisfy the requirement of the BPR, a dossier of an active substance for inclusion 
in  the  Union  list  (or  in  Annex  I  of  active  substances  referred  to  in  Article  25a  of  the  BPR) 
may  be  accompanied  by  such  a  product  as  the  associated  biocidal  product.  To  the  extent 
possible, data from real products are nevertheless recommended. 

While some dummy products may be very similar to a fully formulated product, others may 
be a very simple formulation that bears little resemblance to the product which will finally be 
placed on the market. The latter may be used where the applicant has limited experience in 
formulating products, for example by applicants who only manufacture active substances.  

At the active substance approval stage, the following should be demonstrated: 

The evaluation should demonstrate that the active substance under evaluation is capable of 
producing an  effect when formulated into a very  simple product (active  substance alone  or 
diluted in a solvent) and to define an application rate, which is consistent with the intended 
use(s) claimed by the applicant, and that can be used for the exposure assessment. 

If  a  dummy  product  is  used,  a  more  restricted  level  of  efficacy  could  be  acceptable  if  an 
appropriate  and  detailed  justification  is  given  by  the  applicant.  However,  the  minimum 
requirements mentioned in section 4.2 have always to be fulfilled. 

4.5 Active substances used in treated materials and treated articles 

Treated articles have been included into the biocides legislation  on 1  September 2013 with 
the  BPR  (Biocidal  Products  Regulation).  This  requires  different  considerations  and  testing 
approaches as compared to the previous legislation, BPD. 

Guidance on treated articles is further addressed in sections 5. 3 and 5.4.6. 

4.5.1 Efficacy assessment for active substance approval 

For biocidal products placed on the market in the EU, the authorisation requirements of the 
BPR apply, including testing efficacy. For treated articles imported into the EU, there is only 
the  active  substance  approval  stage  to  test  efficacy.  In  this  respect,  it  is  particularly 
important  to  evaluate  and  assess  use  in  treated  articles  at  the  active  substance  approval 
stage. 

Where  claims  to  treat  articles  are  made  for  active  substance  or  biocidal  products,  efficacy 
data to support these claims have to be submitted (see Annex II, Title 1, 6.6 and Annex III, 
Title  1,  6.6  and  6.7).  If  claims  are  made  on  active  substance  level,  efficacy  assessment  of 
the use in treated articles has to be part of the active substance evaluation. 

4.5.2 Efficacy assessment for active substances in specific PTs 

For  active  substances  notified  for  certain  PTs  it  is  obvious  that  they  are  mainly,  or 
exclusively used, to treat articles/materials as for example for PTs 6, 7, 8, 9, 10 (Main group 
2). Thus, efficacy testing with respect to use to treat articles/materials, is a natural part of 
the  active  substance  evaluation.  In  such  cases  use  concentrations  and  standard  use 

 
   
 
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conditions for use in treated articles have to be taken into account in assessing efficacy. The 
biocidal  function  of  the  PTs  within  Main  group  2  is  usually  protection  of  specific  materials 
from biodeterioration, in some cases odour prevention. The state of the articles treated can 
be solid or liquid. The use conditions can be dry, humid or wet, which can be quite crucial for 
the  release  of  the  active  substance  out  of  the  matrix.  Thus,  the  representative  product 
should  show  the  claimed  effect(s)  in  the  range  of  uses  and  use  conditions  which  are 
described and in the type of matrixes applied for. Use conditions like ageing, weathering or 
washing  should  be  simulated  as  appropriate,  to  demonstrate  the  duration  of  the  effect  in 
relation to the life-span of the article treated. 

Active  substances  notified  for  PTs  1-5  (Main  group  1)  are  usually  used  in  (liquid)  biocidal 
products  as  for  instance  hand  disinfection  or  surface  disinfection  products.  These  products 
are clearly considered biocidal products. But sometimes active substances belonging to PTs 
2,  3  or  4  are  incorporated  into  textiles  and  other  solid  materials;  the  protection  of  the 
material  itself  is  not  intended,  but  a  new  property  is  introduced  to  an  article,  intended  to 
protect  its  user.  For  such  claims,  testing  is  particularly  challenging  and  the  specific 
conditions  of  use  have  to  be  considered  when  designing  the  efficacy  testing.  Please  read 
more  about  how  to  design  such  tests  in  section  5.4.6.  At  active  substance  level,  the 
representative  product  should  show  the  claimed  effect(s)  in  a  range  of  uses  and  use 
conditions  which are described and in the type of matrixes applied for. Particularly the wet 
state  of  the  use  conditions  (dry,  humid  or  wet)  needs  to  be  taken  into  account,  as  this  is 
crucial for the release of the active substance out of the matrix and thus for the efficacy of 
the representative product. Furthermore, use conditions like ageing, weathering or washing 
should be simulated as appropriate, to demonstrate the duration of the effect in relation to 
the life-span of the article treated. Use conditions for which no efficacy of the representative 
product could be demonstrated must be excluded from the approval as appropriate. 

Active  substances  belonging  to  PTs  18  and  19  and  used  to  treat  (solid)  articles  can  have 
different  purposes.  The  treatment  can  be  intended  to  protect  the  material  (for  instance  a 
carpet treated with an insecticide to prevent moth damage) or it can be intended to protect 
humans or animals against insects (for instance clothes treated with a repellent). Again, in 
the latter case it has to be carefully considered whether such a product fulfils the definition 
of a biocidal product and has to undergo an authorisation procedure. At the active substance 
approval stage, any claims made should be demonstrated with appropriate efficacy tests on 
the  representative  product,  taking  into  account  the  specific  conditions  of  use  (e.g.  regular 
washing  for  clothes)  and  the  availability  of  the  active  substance  to  the  target  organisms, 
which can differ in different matrices. 

 
 
 
 
 
   
 
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5. Product authorisation 

5.1 Evaluation of efficacy at product authorisation stage 

The Product Authorisation stage is the point in the evaluation process where the efficacy of 
the  biocidal  product  should  be  looked  at  for  the  full  range  of  claims  made.  More  test 
organisms  or  different  uses  can  be  relevant  as  compared  to  active  substance  approval.  At 
this stage, it is not the properties of the active substance which are of interest, but instead 
the  properties  of  the  fully  formulated  product,  which  may  contain  more  than  one  active 
substance. 

Therefore,  this  is  the  stage  at  which  a  full  evaluation  of  the  efficacy  of  the  formulated 
product  should  be  carried  out,  and  where  the  efficacy  is  evaluated  in  relation  to  the  label 
claims  made  for  the  product.  This  evaluation  should  include  all  relevant  target  species  (or 
representative  species),  the  effects  of  using  the  product,  the  duration  and  speed  of  effect 
(including  ageing  and  weathering  if  relevant),  any  claims  for  residual  action,  together  with 
any other specific claims. 

At biocidal product authorisation, the applicant must clearly describe the uses for which the 
product is intended when it is used under normal conditions, at the  appropriate application 
rate and in accordance with the use instructions. 

This  information  is  required  to  allow  a  proper  evaluation  of  the  efficacy  to  be  carried  out, 
and must include, for every product type separately: 

•  The  purpose  of  the  biocide  (e.g.  prevent  destruction  of  material  by  insect 

infestations, decrease of bacterial contamination on surfaces); 

•  The function of the product (e.g. bactericide, fungicide, rodenticide, insecticide);  

•  The organism(s) to be controlled; 

•  The effects on representative target organism(s) (e.g. attracting, killing, inhibiting); 

•  Any products, organisms or objects to be protected; 

•  The concentration at which the active substance will be used (the use concentrations 
for  different  targets  should  be  stated  for  each  use  and  method  of  application,  if 
appropriate.  Applicants  should  also  indicate  if  the  use  concentrations  should  be 
different in different parts of EU); 

•  Description of the instructions of uses. 

At the product authorisation stage, efficacy must be demonstrated against all claimed target 
organisms.  Use  against  additional  target  organisms  (i.e.  which  were  not  supported  at  the 
active substance approval stage) may be applied for at this stage. 

For biocidal products used to treat articles, it is important to categorise possible wide ranges 
of uses into sets of similar materials and use-conditions. Please see sections 5.3, 5.4.2 and 
5.5 for more details. 

5.2 Product families 

5.2.1 Background 

A product family is a group of products with the same active substance(s) and similar use, 
but small differences in the formulation, which do not significantly reduce the efficacy of the 

 
   
 
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products.2.  When  authorisation  is  requested  for  a  product  family  efficacy  should  be 
demonstrated for the whole group but not necessarily of each product. A product family can 
be divided in different meta SPC’s3, and all products in the meta SPC have the same hazard 
and precautionary statements. However, it is also possible that extra  meta SPC's should be 
added  because  of  the  efficacy  assessment  (e.g.  some  products  in  the  family  are  not 
efficacious  for  some  uses).  It  should  thus  be  noted  that  the  efficacy  evaluation  of  the 
product  family  should  be  made  in  conjunction  with  the  other  parts  of  the  evaluation  (e.g. 
ENV,  HH  and  phys-chem)  and  that  an  overall  assessment  of  the  division  into  meta  SPC’s 
should  be  made  taking  all  areas  into  account.  This  guidance  is  specifically  aimed  at  an 
evaluation of differences in efficacy claim, which could lead to certain structures of the BPF 
and meta SPC’s. Therefore, some of the following examples could result in other structures 
of the meta SPC’s when environment, human health and phys-chem are taken into account. 

5.2.2 Worst case testing 

The  BPF  concept  allows  read-across  of  data  between  similar  products  within  and  across 
meta SPCs. Efficacy tests must be performed on the product with the lowest concentration of 
the  active  substance,  under  the  worst  case  circumstances.  The  influence  of  the  co-
formulants  on  the  efficacy  should  be  taken  into  account.  A  justification  should  be  given  for 
the product and circumstances taken. 

Tests  and  criteria  for  testing  efficacy  of  products  in  a  family  are  the  same  as  for  single 
products. For the data requirements and test criteria, please see the specific sections per PT. 

Applicants need to ensure that all products within a family have been supported, in terms of: 

• 

• 

• 

• 

target organisms;  

concentrations / application rates;  

contact time; 

influence of the co-formulants; 

•  application methods;  

• 

field of use / use conditions; 

•  other label claims;  

• 

formulations; 

•  any other relevant information.  

2 See Article 3 of the BPR for the full definition of a BPF. 

3 See for the definition of a meta SPC CA-Nov15- 
Doc_4_3Update_note_for_guidance_on_BPF_concept.docx 

 
 
 
 
 
   
 
 
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Table 1: Example ready-to-use disinfectants with/without pre-cleaning*. 

Family A 
Concentration AS: 1-4% 

meta SPC 1 

meta SPC 2 

Product 1 

Product 2 

Product 3 

concentration AS 

1% 

target organisms 

bacteria 
yeasts 

1% 

bacteria 
yeasts 

4% 

Bacteria 
yeasts 
viruses 

use conditions 

apply after pre-
cleaning 

apply after pre-
cleaning 

apply without cleaning 

colour 

1 

2 

1 

NOTES to Table 1 

In  this  example  one  worst  case  for  efficacy  cannot  be  identified.  Product  1  should  be  tested 
against  bacteria  and  yeasts  under  clean  conditions  (also  supporting  product  2),  and  product  3 
should be tested against bacteria, yeasts viruses, under dirty conditions. 

Since  these  are  all  ready-to-use  products,  and  presuming  that  1%  is  not  efficacious  against 
viruses,  product  1  and  2  should  be  in  a  different  meta  SPC  than  product  3  since  they  are  not 
efficacious  against  viruses.  The  meta  SPC  of  products  1  and  2  will  state  as  target  organisms 
bacteria and yeasts and the meta SPC of product 3 bacteria, yeasts and viruses. 

* In the examples, only the information given in the table is taken into account for the deviation in 
meta  SPC’s,  presuming  that  all  other  factors  are  the  same  for  the  different  products  or  of  no 
influence. In practice other factors relating to the products will also need to be taken into account. 

In  some  cases  it  is  not  possible  to  identify  one  worst  case  scenario  for  a  combination  of 
products  and  use  conditions:  where  such  a  single  “worst  case”  scenario  at  meta  SPC  level 
cannot  be  identified,  an  assessment  of  the  minimum  efficacy  levels  that  might  be  relevant 
for the uses covered by a meta SPC has to be performed. For instance, the family contains 
products  (1)  and  (2)  with  low  active  substance  (AS)  concentration  which  will  be  used  as 
disinfectant  under  clean  conditions  and  only  for  the  control  of  bacteria  and  yeast,  while 
another product (3) with a higher concentration of AS is used under dirty conditions for the 
control of bacteria, yeast, and viruses. Product (1) and (2) will not be sufficiently efficacious 
against viruses, so it cannot be used to demonstrate efficacy for all the uses. In this family, 
product  (1)  should  be  tested  under  clean  conditions  against  bacteria  and  yeast  (and  cover 
product  (2))  and  product  (3)  should  be  tested  under  dirty  conditions  against  bacteria  and 
yeast  and  viruses  (see  Table  1).  Tests  done  for  a  product  in  one  meta  SPC  can,  where 
relevant, be used to support a claim for a similar product in a different  meta SPC, provided 
that variations in  co-formulants have no influence  on efficacy. Justification may need to be 
provided to allow read across. 

In  some  product  families  several  combinations  of  products  and  uses  should  be  tested,  to 
demonstrate  efficacy  for  all  combinations  of  products  and  use  conditions  (see  Tables  2,  3, 
and 4). 

 
   
 
 
 
 
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Family B 
Concentration AS: 10-40% 

meta SPC 
Product: 10-40% AS 
Dilute product to use concentration: 
bacteria: 1% AS 
fungi: 1% AS 
viruses: 4% AS 

Product 1 

Product 2 

Product 3 

concentration AS 

target organisms 

10% 

bacteria 
fungi 

20% 

bacteria 
fungi 

40% 

Bacteria 
fungi 
viruses 

NOTES to Table 2 

In  this  example  all  products  are  concentrates  to  be  diluted  before use.  The  applicant  only  claims 
efficacy  against  bacteria  and  fungi  for  product  1  and  2  and  in  addition  viruses  for  product  3. 
Presuming all products only differ in the concentration active substance, testing can be done with 
either  of  the  products  at  use  concentration:  product  diluted  to  1%  active  substance  should  be 
tested  against  bacteria  and  fungi,  and  product  diluted  to  4%  active  substance  should  be  tested 
against viruses. 

Since all concentrated products can be diluted to an efficacious concentration, when used according 
to the instructions on the meta SPC, all products can be in one meta SPC. 

Table 3: Example surface disinfectants ready-to-use: more PT’s 

Option 1 

Option 2 

Family C 
Concentration AS: 10% 

meta SPC 1 
Use #1: PT3, bacteria, fungi 
Use #2: PT4, bacteria, fungi, viruses 

meta SPC 1 
Use #1:  
PT3, bacteria, fungi 

meta SPC 2 
Use #2:  
PT4, bacteria, fungi, 
viruses 

Product 1 

Product 2 

Product 3 

concentration AS 

target organisms 

10% 

bacteria 
fungi 

10% 

bacteria 
fungi 

PT 

PT3 

PT3 

10% 

Bacteria 
fungi 
viruses 

PT4 

NOTES to Table 3 

In this example all products are ready to use and have the same use concentration, they only have 
a different use claim (i.e. same use in different PTs). It is presumed that the products only slightly 
differ in their composition  and that it is demonstrated that this does not influence  the efficacy. In 

 
 
 
 
 
   
 
 
 
 
 
 
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this case either of  the products can be tested under  worst  case conditions (justification should be 
given  that  PT3  soiling  and  temperature  is  the  worst  case).  A  representative  product  should  be 
tested against the specified bacteria and fungi required for PT3, and against the specified bacteria 
and viruses required for PT4. Since the fungi that have to be tested for PT3 and PT4 are identical, 
one test performed under  the worst  case  conditions  is sufficient. Since this  meta  SPC can be split 
into  2  uses,  one  for  PT3  and  one  for  PT4,  and  all  products  are  efficacious  against  all  uses,  it  is 
possible  to  put  all  three  products  in  one  meta  SPC,  (option  1).  All  possible  products  in  this  meta 
SPC  will  be  efficacious  against  use  #1  and  use  #2.  Efficacy  against  viruses  in  PT3  is  not 
demonstrated, however, since this is not in one of the uses in the meta SPC, this is acceptable. On 
the product label only the specified uses, combination of PT and target organisms, can be claimed. 
However,  an  applicant  might  consider  it  easier  to  split  the  family  in  2  meta  SPC’s  ,  one  per  PT 
(option 2). 

Table 4: Example insecticide: take target organisms and application method into 
account. 

Family D 
Concentration AS: 1-4% 

meta SPC 1 
Conc. AS: 1% 

meta SPC 2 
Conc. AS: 1% 

meta SPC 3 
Conc. AS: 4% 

Product 1 

Product 2 

Product 3 

concentration AS 

target organisms 

1% 

moth 

application method 

paper in wardrobe 

1% 

moth and mosquitoes 

electric device in 
wardrobe or room 

4% 

Ants 

bait box with sugar 

NOTES to Table 4 

In this example one worst case for efficacy testing cannot be identified and all products should be 
tested for all target organisms and uses.  

All  three  products  should  be  in  different  meta  SPC’s  because  of  the  different  application  methods 
and organisms. 

When a family contains more than one active substance it might not be sufficient to test the 
products  to  be  authorised  in  a  meta  SPC,  in  some  cases  it  is  necessary  to  test  a  ‘dummy’ 
product  to  cover  all  products  in  one  meta  SPC  (see  Table  6).  Alternatively,  they  could  be 
authorised in separate meta SPC. 

5.2.3 Take formulation types and chemical composition into account 

While  the  active  substance  is  the  most  important  constituent  for  efficacy  of  a  biocidal 
product, the effect of the formulation of the product on the efficacy must also be taken into 
account. Therefore, the justification should be given for the product used in the test, taking 
into  account  the  formulation.  If  the  product  contains  more  than  one  active  substance,  the 
combined effect between different active substances will be considered. 

In the case of products having different formulation types (e.g.  wettable powder and water 
dispersible  granules  for  PT18),  bridging  studies  with  these  products  can  be  used  to 
substantiate  that  the  products  are  equivalent  in  terms  of  their  efficacy.  Bridging  studies 
should involve worst case circumstances (after appropriate justification). 

Depending on the influence of the ingredients (chemical composition) on the efficacy either 
the product with the lowest concentration of all the ingredients should be tested or several 

 
   
 
 
 
 
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products, together including the whole spectrum of the formulations, should be tested (see 
Table 5). 

5.2.4 Allowing for the addition of new products in a family 

In general the (meta) SPC(s) of a family will give a range for the concentration of the active 
substance(s)  and  co-formulants.  After  authorisation  of  the  family  it  is  possible  to  add  new 
products to the family, as long as their composition falls into the range for the (meta) SPC. 
For  these  new  products  no  evaluation  will  be  done.  Therefore,  efficacy  testing  should  be 
done in such a way that efficacy against all possible new products will be demonstrated. 

For instance, in the example in Table 5, a new product with 70% active substance and the 
lowest  concentration  of  both  acids  could  be  added.  Efficacy  of  this  product  should  be 
demonstrated, or the two products should be put into different meta SPCs. Another example 
is explained in Table 6. 

Table 5: Example disinfectant: take formulation into account. 

Family E 
Concentration AS: 70-85% 
Concentration acid 1: 1-4% 
Concentration acid 2: 2-5% 

Option 1 

meta SPC 1 
Concentration AS: 70-75% 
Concentration acid 1: 1-4% 
Concentration acid 2: 2-5% 

meta SPC 2 

Option 2 

meta SPC 1 

meta SPC 2 

meta SPC 3 

Product 1 

Product 2 

Product 3 

target organisms 

Active substance 

Acid 1 

Acid 2 

NOTES to Table 5: 

bacteria 
fungi 

70% 

1% 

5% 

bacteria 
fungi 

75% 

4% 

2% 

bacteria  
fungi 
virus 

85% 

1% 

5% 

In  this  example  both  acids  are  pH  regulators.  It  is  presumed  they  are  not  considered  active 
substances in this formulation (in some cases this should be demonstrated with tests), however, 
both acids might enhance the efficacy to some extent (i.e. formulation effect). Since it cannot be 
ruled out that there is a difference in effect between these two acids, this should be taken into 
account in the efficacy testing. 

When product 1 and 2 are placed in one  meta SPC (option 1) it should be considered that it is 
possible to add a new product in this meta SPC with 1% acid 1 and 2% acid 2. In that case it is 
not sufficient to test product 1 (with lowest concentration AS), but a ‘dummy’ product should be 
tested, with 70% AS, 1% acid 1 and 2% acid 2. 

To  prevent  testing  with  ‘dummy’  products,  it  might  be  easier  to  place  products  1  and  2  in 
separate  meta  SPC’s,  without  a  range  for  the  acids  (option  2).  Also  in  that  case,  read  across 
between product 1 and 2 is not possible. Both product 1 and 2 should be tested, to rule out the 
effect of the formulation with different acid concentrations. 

 
 
 
 
 
   
 
 
 
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In all cases product 3 should be tested against viruses, and put in a different meta SPC 
(assuming 85% is necessary for viruses). The test with product 1 or the ‘dummy’ product can be 
used to demonstrate efficacy against bacteria and fungi for meta SPC 2 (product 3). 

5.2.5 Deviation in meta SPC’s 

When  dividing  a  product  family  in  meta  SPC’s,  it  must  be  taken  into  account  that  all 
(possible  new)  products  will  be  efficacious  for  all  uses,  target  organisms,  etc.  Worst  case 
testing  must  make  sure  that  all  possible  new  products  will  be  efficacious.  Where 
needed/possible new meta SPC’s should be made for a different group of target organisms, 
a different use, different application method, etc.  

This means for the example family in Table 4, that all products should be in a different meta 
SPC.  

In  Table  1  product  1  and  2  should  be  separated  from  product  3,  because  these  are  not 
efficacious against viruses and therefore not against all target organisms in this meta SPC.  

However, in some cases it might be possible to not deviate in more  meta SPC’s but give a 
good  description  in  the  meta  SPC,  making  sure  that  all  products  will  be  efficacious.  For 
instance, in the examples in Tables 2 and 3, which are very similar to Table 1, the product 
with  a  virus  claim  can  be  in  the  same  meta  SPC.  This  is  acceptable  because  all  possible 
products are efficacious when used according to the use description in the  meta SPC, either 
because  all  products  can  be  diluted  to  an  efficacious  dose,  or  by  making  separate  use 
numbers.  In  these  cases  some  of  the  products  in  the  meta  SPC  have  a  limited  claim  (i.e. 
fewer organisms, fewer PT’s). 

When  the  different  uses  results  in  a  too  complicated  meta  SPC,  with  several  different  use 
numbers, it is better to divide such a meta SPC in more simpler meta SPC’s. 

When dividing into meta SPC’s the applicant must make sure that the text in the meta SPC’s 
is  unambiguous,  and  consider  that  no  products  can  be  added  to  the  family  that  have  not 
been supported in the efficacy testing (see Tables 3 and 4). 

Table 6: Example anti-fouling product: Different ratios of two (or more) active 
substances. 

Option 1 

Option 2 

Family 
Concentration.AS 1: 5-10% 
Concentration.AS 2: 2-7% 

meta SPC 1 
Concentration.AS 1: 5-10% 
Concentration.AS 2: 2-7% 

meta SPC 1 
Conc. AS 1: 10% 
Conc. AS 2: 2% 

meta SPC 2 
Conc. AS 1: 5% 
Conc. AS 2: 7% 

Product 1 RTU 

Product 2 RTU 

target organisms 

Macro fouling 

Macro fouling 

Active substance 1 

Active substance 2 

10% 

2% 

5% 

7% 

 
   
 
 
 
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NOTES to Table 6: 

In this example testing product 1 and 2 is not sufficient to cover the worst-case situation of this 
family.  The  worst-case  would  be  a  product  5%  active  substance  1  +  2%  active  substance  2  . 
Assuming variation of co-formulants have no impact on efficacy, this  ‘dummy’ product should be 
tested  to  demonstrate  efficacy  for  this  family  when  it  consists  of  one  meta  SPC  (option  1). 
Alternatively,  product  1  and  2  can  be  put  into  different  meta  SPC  (option  2),  and  efficacy  test 
using prod 1 and 2 can be provided. 

5.2.6 Minimum concentration needed 

Whilst ready-to-use products authorised on their own are evaluated on their merits and not 
in comparison to other products, this is not the case in a product family. Since all products 
are presented at the same time a comparison can be made. The BPR Annex VI art. 77 of the 
common principles state: the recommended dose is the minimum necessary to achieve the 
desired effect. 

For historical reasons it is possible that products on the market in one EU country contain a 
higher  concentration  of  AS  than  another  product  with  the  same  intended  use  in  another 
country. When this is the case the applicant should request for authorisation for the products 
with  the  lowest  concentration  of  AS  or  give  a  good  justification  why  it  is  relevant  to  have 
different formulations. 

It  should  be  considered  that  there  may  be  other  products  on  the  market  which  contains  a 
lower concentration of AS and is efficacious for the same intended use. 

5.3 Treated articles 

NOTE to the reader:  
This section concerns treated articles and should be read in conjunction with the CA 
Note  for  Guidance  “Frequently  asked  questions  on  treated  articles”,  CA-Sept13-
Doc.5.1.e, Revision 1 December 2014 4. 

Article 3 Definitions 

1. For the purposes of this Regulation, the following definitions shall apply: 

(a) ‘biocidal product’ means 

- any substance  or mixture, in the form in which it is supplied to the  user, consisting 
of,  containing  or  generating  one  or  more  active  substances,  with  the  intention  of 
destroying, deterring, rendering harmless, preventing the action of, or otherwise exerting a 
controlling  effect  on,  any  harmful  organism  by  any  means  other  than  mere  physical  or 
mechanical action, 

-  any  substance  or  mixture,  generated  from  substances  or  mixtures  which  do  not 
themselves  fall  under  the  first  indent,  to  be  used  with  the  intention  of  destroying, 
deterring, rendering harmless, preventing the action of, or otherwise exerting a controlling 
effect  on,  any  harmful  organism  by  any  means  other  than  mere  physical  or  mechanical 
action. 

A treated article that has a primary biocidal function shall be considered a biocidal product. 

(l) ‘treated article’ means any substance, mixture or article which has been treated with, or 
intentionally incorporates, one or more biocidal products. 

4 CA-Sept13-Doc 

 
 
 
 
 
   
 
 
 
 
 
 
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A treated article according to Article 3(1)(l) of  the BPR is any substance, mixture  or article 
which  has  been  treated  with  or  intentionally  incorporates  one  or  more  biocidal  products.  A 
biocidal product, in contrast, is any substance or mixture with a biocidal  function. Pursuant 
to  Article  3(1)(a)  a  treated  article  with  a  primary  biocidal  function  is  considered  a  biocidal 
product. 

Liquids  fulfil  the  substance  or  mixture  definition.  Consequently,  liquids  may  only  be 
considered  as  treated  articles  if  they  do  not  intend  to  control  any  harmful  organism.  In 
contrast,  solid  treated  articles  are  defined  by  their  shape  and  function  rather  than  by  their 
chemical composition. Thus, solid treated articles fulfil the definition of a biocidal product if 
they have a primary biocidal function. 

The  term  “primary  biocidal  function”  is  not  further  defined  in  the  BPR,  but  in  the  CA 
document, it is described as “a biocidal function of first rank, importance, or value compared 
to other functions of the treated article”. 

A  biocidal  product,  in  contrast,  is  any  substance  or  mixture  with  a  biocidal  function. 
Consequently,  efficacy  testing  and  assessment  is  not  principally  different  for  biocidal 
products and treated articles. Both categories can take different forms (liquid, solid) and can 
concern different materials. In both cases efficacy has to be shown for normal conditions of 
use  and  against  an  untreated  control.  The  untreated  control  should  demonstrate  the 
problem which is to be solved by the biocidal treatment. 

Thus, considering the different product types for PTs 1-4, the following examples would be 
considered  as  biocidal  products  and  not  treated  articles.  For  PT  1  or  3,  disinfecting  wipes 
would  be  regarded  as  biocidal  products5.  For  PT2,  paints  and  coatings  intended  to  prevent 
microbial settlement and growth in order to provide a hygienic  environment would likewise 
be  regarded  as  biocidal  products6.  Other  PT  2  applications  which  could  fall  under  either 
category,  depending  on  their  primary  function  could  include  for  instance  textiles,  tissues, 
masks, or other articles or materials in which a biocidal product has been incorporated with 
the  purpose  of  adding  disinfecting  properties  to  these  articles  and  materials.  For  PT  4, 
examples are materials or articles which come into contact with food or feed and are treated 
with or incorporate a biocide; whether such articles are to be regarded as biocidal products 
again  depends  on  their  primary  function.  PT  5  applications  are  usually  biocidal  products. 
Further product examples are given in Appendix 1 of the CA document. 

There are some exemptions in the definition given in Art. 3(1)(a): Articles such as paper or 
carton, where the pulp has been treated with a biocide during manufacture, and where the 
biocide  is  not  intended  to  have  a  function  in  the  final  good  are  not  considered  treated 
articles. Another example are articles with print on it or with glue holding it together which 
have been treated with an in-can preservative. However, the preservative doesn’t have any 
function in the final article as soon as the ink or adhesive is applied and dried. In contrast, 
an  article  like  a  table  made  of  a  composite  material  with  wooden  legs  painted  with  a  film 
preservative  containing  coating,  is  considered  a  treated  article,  as  the  coating  still  has  a 
biocidal function in the final article. 

Generally, there is no difference in efficacy testing of treated articles or biocidal products in 
a liquid matrix. For instance, wet state preservatives (PT 6) or a hand disinfectant (PT 1) are 
usually  both  tested  in  a  liquid  matrix,  the  first  matrix  is  a  treated  article,  the  latter  is  a 

5 See CA document Appendix 1 

6 See CA document Question 8 

 
   
 
 
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biocidal  product;  only  the  performance  standards  are  different  in  these  examples.  Specific 
requirements apply, however, when the efficacy of solid material or articles has to be tested. 
A test under practical conditions of use (step 3 test) is mandatory. In contrast to preserving 
claims,  where  standard  materials  under  certain  standard  conditions  of  use  can  be  tested, 
testing  for  disinfecting  claims  has  to  be  specific  for  every  single  article.  For  these  types  of 
claims,  the  specific  conditions  of  use  are  to  be  considered  when  designing  the  efficacy 
testing; for example, a polymer coating used for a hospital bedside cabinet has to be tested 
for  the  specific  contaminating  situation  of  a  hospital  bedside  cabinet,  including  cleaning 
schemes and  soiling  situation; efficacy has to  be  shown  compared to  an untreated bedside 
cabinet. Bactericidal effects have to take effect very quickly to show an advantage compared 
to an untreated cabinet, where droplets of blood or saliva will dry out quickly and not either 
be contaminating any more. Please read more about how to design such tests in Section5.3. 

Specific  requirements  apply,  however,  when  the  efficacy  of  biocides  in  solid  material  or 
articles has to be tested. Treated articles with claims to protect humans or animals fall under 
this  category.  In  these  cases,  use  conditions,  most  importantly  humidity,  have  to  be 
specified.  Materials  can  be  used  in  articles  with  a  wide  range  of  use  conditions,  and  these 
have an effect on efficacy. For example, for a polymer article permanently exposed to water 
the  conditions  for  bacterial  growth  are  much  more  favourable,  and  different  requirements 
apply  as  compared  to  a  polymer  article  which  is  generally  dry  and  is  only  exposed  to 
occasional splashes or to the humidity which comes from touching it. But more importantly, 
humidity  has  an  effect  on  the  availability  of  the  active  substance,  because  it  has  to  be 
released  out  of  the  matrix  somehow.  Another  example  are  clothes  treated  with  repellents; 
also in this case use-conditions do influence efficacy. Wearing and tearing and washing have 
to  be  taken  into  account  to  assess  the  efficacy.  Complete  protection  time  needs  to  be 
defined in terms of the life-cycle of the treated clothes. 

Treated articles, if not biocidal products, do not require efficacy assessment under the BPR. 
However,  active  substances  and  biocidal  products  incorporated  into  treated  articles  may 
require  assessment  of  their  efficacy  in  treated  articles  as  part  of  the  active  substance 
approval and biocidal product authorisation processes (if such uses are applied for). 

Consequently,  if  efficacy  is  demonstrated  for  a  certain  set  of  use  conditions,  this  cannot 
generally  be  transferred  to  another  set  of  use  conditions.  The  possible  limits  of  the  use 
conditions  have  to  be  reflected  in  the  approval/authorisation  decision.  In  the  following, 
guidance  is  given  for  the  testing  of  (solid)  materials  with  claims  to  protect  humans  or 
animals. 

There are two OECD test methods available: 

•  Guidance Document on the Evaluation of the Efficacy of Antimicrobial Treated Articles 

with Claims for External Effects (OECD Series on Biocides No. 1); 

•  Guidance  Document  for  Quantitative  Method  for  Evaluating  Antibacterial  Activity  of 
Porous and Non-Porous Antibacterial Treated  Materials (OECD  Series on Testing and 
Assessment No. 202 and Series on Biocides No. 8). 

5.3.1 The basic distinction between material protection and protection of 
humans or animals 

When  biocides  are  incorporated  into  materials  or  used  in  the  production  of  treated  articles 
they are applied with two purposes: 

 
 
 
 
 
   
 
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•  To protect the materials used in the article or the properties of the article in service. 
The  target  organisms  have  a  detrimental  or  other  undesirable  effects  (e.g. 
biodegradation, discolouration, odour formation) on the material or article. 

•  To  protect  humans  or  animals  from  the  unwanted  effects  of  organisms.  The 
treatment is directed towards targets organisms which have no adverse effect on the 
item/material treated. 

The following scheme gives an overview and decision help: 

Figure  1:  Decision  scheme  to  distinguish  between  claims  for  material  protection 
and claims for protection of humans and animals 

Is the treatment intended to 
protect the material, article or its 
functionality from biological 
deterioration in service, extend its 
durability or prevent odour? 

Yes 

No 

Main Group 2, Main group 3 
(PT 18, 19) of Annex V BPR 
________________________ 
Protection of material/article 
and its properties; 
sections 5.5 and specifically 
5.5.7-5.5.9 

Main Group 1 (PT 1-5), Main 
group 3 (PT 18, 19) of Annex V 
BPR 
________________________ 
Adds properties to protect 
humans or animals; 
section 5.4.6 

Inhibits Growth 
_____________ 
section 5.4.6.2 

Kills, Repels 
_____________ 
section 5.4.6.3  

Guidance  for  the  testing  of  biocidal  products  with  a  claim  to  protect  humans  or  animals  is 
given in section 5.4.6. Guidance for material protection is given in section 5.5. 

 
   
 
 
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5.4 Disinfectants (Main group 1) 

5.4.0 General 

5.4.0.1 Introduction 

This guidance describes the nature and extent of data which should be available to support 
the  label  claims  for  biocidal  products  within  the  Main  Group  1:  Disinfectants.  This  group 
covers 5 product types as described in Annex V of the BPR: 

MAIN GROUP 1: Disinfectants 

These  product-types  exclude  cleaning  products  that  are  not  intended  to  have  a  biocidal 
effect, including washing liquids, powders and similar products. 

Product type 1:   Human hygiene  
Products in this group are biocidal products used for human hygiene purposes, applied on or 
in  contact  with  human  skin  or  scalps  for  the  primary  purpose  of  disinfecting  the  skin  or 
scalp. 

Product type 2:   Disinfectants  and  algaecides  not  intended  for  direct  application 
to humans or animals  
Products used for the disinfection of surfaces, materials, equipment and furniture which are 
not used for direct contact with food or feeding stuffs. 

Usage areas include, inter alia,  swimming pools, aquariums, bathing and other waters; air-
conditioning  systems;  and  walls  and  floors  in  private,  public,  and  industrial  areas;  and  in 
other areas for professional activities. 

Products  used  for  disinfection  of  air7,  water  not  used  for  human  or  animal  consumption, 
chemical toilets, waste water, hospital waste and soil. 

Products used as algaecides for treatment  of  swimming pools, aquariums and other waters 
and for remedial treatment of construction materials. 

Products  used  to  be  incorporated  in  textiles,  tissues,  masks,  paints  and  other  articles  or 
materials with the purpose of producing treated articles with disinfecting properties. 

Product type 3:   Veterinary hygiene 
Products used for veterinary hygiene purposes such as disinfectants, disinfecting soaps, oral 
or corporal hygiene products or with anti-microbial function. 

Products  used  to  disinfect  the  materials  and  surfaces  associated  with  the  housing  or 
transportation of animals 

Product type 4:   Food and feed area 
Products  used  for  the  disinfection  of  equipment,  containers,  consumption  utensils,  surfaces 
or  pipework  associated  with  the  production,  transport,  storage  or  consumption  of  food  or 
feed (including drinking water) for humans and animals. 

7 This is taken to mean the disinfection of air itself. Disinfectants sprayed or vaporised into the air (e.g. 
room  disinfection  by  vaporised  biocide)  are  normally  for the purpose  of disinfecting surfaces  and not 
the  air  itself.    Disinfectants  for  air  conditioning  systems  disinfect  the  surfaces  or  liquids  in  these 
systems, not the air coming out of it. 

 
 
 
 
 
   
 
 
 
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Product type 5:   Drinking water 
Products used for the disinfection of drinking water for both humans and animals. 

Products in this main group are meant for the control of micro-organisms, such as bacteria 
(including  vegetative  cells,  spores  and  mycobacteria),  fungi  (including  moulds  and  yeasts), 
and  viruses  (including  bacteriophages),  algae  and  protozoa.  Control  may  be  carried  out  on 
inanimate  surfaces  or  skin  or  in  liquids.  Note  that  the  term  "disinfectant"  used  for  main 
group 1 should be read as a generic term and not according to the definition in the glossary 
of  terms.  This  means  that  next  to  disinfectants  it  can  also  include  products  with  biostatic 
activity. 

The most important fields of use include medical, veterinary, food, feed and drinking water 
sectors.  Applications  in  public,  commercial  and  industrial  areas,  where  application  is  to 
inanimate  surfaces  without  direct  contact  with  food,  are  included  in  Product  type  2.  If 
contact  between  disinfected  inanimate  surfaces  and  food  is  possible  (e.g.  food  industry, 
private and restaurant kitchens), applications are included in Product type 4. 

Disinfectants  for  medical  instruments  and  medical  equipment  that  are  considered  medical 
devices  are  covered  under  the  Medical  Device  Directive  93/42/EEC  (see  3.9.1  for  more 
information). More borderline cases with other Directives or Regulations are noted elsewhere 
in this Guidance Document and are defined or described in other legislation or guidance. 

Cleaning  products  which  are  not  intended  as  biocides,  including  liquid  detergents,  washing 
powders etc. are excluded from these product types and thus this guidance is not applicable 
(Annex V of BPR). 

Treated articles with claimed disinfecting properties or function can also fall within PTs 1 to 
5: when such articles have a primary biocidal function they are considered biocidal products 
(see  Competent  Authority  (CA)  document  8).  These  articles  can  include  a  wide  variety  of 
goods,  with  different  applications,  matrices  etc.  This  guidance  deals  mainly  with  efficacy 
testing of (liquid) biocidal products; the methodology for testing (solid) treated articles can 
be quite different. See section 5.4.4.3 of this Guidance for details of available guidance. 

A “Glossary of Terms” is at the beginning of the document. 

5.4.0.2 Dossier requirements 

The  following  aspects  are  relevant  for  the  evaluation  of  the  efficacy  of  biocidal  products 
within PT1-5: 

1.  The label claim and instructions for use 

2.  Efficacy data of the product 

3.  The possible occurrence of resistance, cross-resistance or tolerance. 

5.4.0.3 Label claim 

For each product, clear label claims should be provided. When the label itself cannot contain 
all  the  necessary  information,  any  accompanying  leaflet  should  also  be  considered.  To 
simplify the text only the term "label claim" will be used below. 

The types  of  efficacy  claims made for a disinfectant/ biocidal product  depend upon, among 
other  things,  the  types  of  micro-organisms  the  disinfectant  targets  (e.g.  fungal  spores, 
yeasts, mycobacteria, bacteria or bacterial spores) and the disinfectant’s intended use (e.g. 

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in  hospitals,  in  contact  with  food,  in  animal  houses,  in  homes).  Label  claims  and 
recommendations  for  use,  including  concentration  and  contact  time,  must  be  supported  by 
the results of bactericidal, fungicidal, etc. tests appropriate to the area of application, which 
are normally performed on the basis of the specific standards. Complete instructions for use 
are an integral part of the label. 

The  information  on  the  product  label  should  fully  correspond  with  the  uses  pre-defined  at 
the  authorisation  stage  and  reflected  in  the  corresponding  version  of  the  SPC9.  Applicants 
must indicate clearly on the product's label the spectrum of antimicrobial activity claimed.  

Examples  of  the  common  fields  of  applications  are  presented  in  the  claims  matrices  which 
are a set of tables linked to this guidance document (see Appendix 1 for more information). 
The Claim Matrices are not intended to be exhaustive, but the majority of uses are included. 

5.4.0.3.1 Target Organisms 

The target organisms for which claims are made should be specified on the product label. 

As  the  claimed  antimicrobial  efficacy  for  disinfectant  products  will  encompass  a  large 
spectrum  of  potential  target  organisms,  it  is  not  necessary  or  indeed  feasible  to  include  all 
possible  micro-organisms  in  an  efficacy  test  designed  to  support  a  label  claim.  Instead  the 
types  of  target  organism  the  product  is  intended  for  are  mentioned,  for  example,  fungal 
spores, yeasts, viruses, algae, protozoa, (myco)bacteria or bacterial spores. 

Specific  species  are  mentioned  on  the  label  where  they  are  the  only  or  most  relevant 
organisms,  or  where  they  have  a  different  susceptibility  to  biocides  than  the  rest  of  the 
group.  For  instance,  mycobacteria  are  less  susceptible  than  other  bacteria  and  it  is  only 
relevant to control them in certain situations such as tuberculosis wards. 

In  general  it  is  not  possible  to  claim  against  specific  single  species  without  claiming  (and 
demonstrating)  efficacy  against  the  group  of  organisms  (e.g.  no  claim  against 
Mycobacterium  tuberculosis  without  also  making  a  general  bactericidal  claim,  no  claims 
against  HIV  without  a  general  claim  against  enveloped  viruses).  However,  there  are  some 
cases in which it can be justified that a claim only for a single or a small number of species 
is  made  (such  as  bacteriophages  in  the  milk  industry,  or  fungi  Aspergillus  fumigatus  in 
poultry housing.). 

Claims against specific organisms or groups of organisms should not be made, if they imply 
a false impression of superiority of a product; for example, a claim against MRSA should not 
be  made  for  a  bactericidal  product,  because  MRSA  do  not  present  a  specific  challenge  for 
disinfectants. 

Standard test methods  normally specify  one or more representative  species that  should be 
tested  per  group  of  organisms  for  which  the  claim  is  made.  For  instance,  a  bactericidal 
product should be tested on gram-positive and gram-negative bacteria, a fungicidal product 
should be tested  on  yeasts and fungal  spores. The  species used are representative species 
that  take  into  account  their  relevance  to  practical  use,  susceptibility  for  disinfectants  and 
adequacy for laboratory testing. 

The  test  organisms  and  strains  which  should  be  used  are  normally  stated  in  standard 
efficacy test methods, i.e. according to EN 14885 or OECD-guidance. 

9 Details on how to fill out the SPC are available in the ECHA Technical Guide and SPC Editor.  

 
 
 
 
 
   
 
 
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When it is not possible to use standard test methods for efficacy testing and other tests are 
used instead, the test organisms listed in Appendix 3 should be employed. If test organisms 
other than those listed in Appendix 3 are used, their relevance should be justified. 

Wherever  possible  strains  should  be  selected  from  international  collections  (their  genetic 
stability  should  be  checked  regularly).  The  preservation  procedures  must  be  clearly 
described (EN 12353). 

Other test organisms, in addition to those specified in the test standards, can also be tested. 
When efficacy against specific additional species is claimed, efficacy tests with those species 
should  also  be  performed.  In  general,  claims  should  not  be  made  against  the  specific 
reference species used in a standard test as this can give a misleading impression that the 
product  shows  activity  beyond  that  covered  by  the  general  (e.g.  bactericidal,  fungicidal) 
claim. 

Mentioning  specific  organisms  on  the  label  is  still  a  subject  of  discussion  between  Member 
States. The above sections reflect the position at the time that this guidance is written. 

For  some  areas  of  use  there  are  minimum  requirements  for  the  groups  of  organisms  for 
which efficacy should be demonstrated. For instance, for products used for animal transport 
vehicles  efficacy  against  bacteria,  yeasts  and  viruses  should  be  demonstrated.  For  these 
products  it  is  obligatory  to  test  all  required  organisms.  Per  section,  a  sub-section  on  test 
organisms provides information on the minimum requirements for that use. 

5.4.0.3.2 Areas of Use 

Disinfectants  are  used  almost  everywhere  that  people  want  to  “eliminate”  or  inhibit  (for 
static  products)  micro-organisms.  They  are  used  to  kill  or  irreversibly  inactivate  or  inhibit 
bacteria,  fungi  and  viruses  on  animate  and  inanimate  surfaces  and  matrices,  in  hospitals, 
households,  schools,  restaurants,  offices,  swimming  pools,  kitchens,  bathrooms,  dairy 
farms, on medical and dental equipment, eating utensils and at many other locations. 

In  some  cases  biostatic  products  are  used  which  only  inhibit  micro-organisms  (see  section 
5.4.0.5.3 of this guidance). 

Applicants should clearly indicate the intended areas of use for the product on the label, for 
example, areas of use could include (not exhaustive): 

•  Hospital and other medical areas; 

•  Domestic use; 

• 

• 

Institutional use (offices, schools etc.); 

Industrial applications, e.g. food, cosmetic, pharmaceutical industry etc.;  

•  Restaurants and large-scale/canteen kitchens; 

•  Veterinary areas (animal housing, animal health care, teat or hoof disinfection etc.); 

•  Recreational areas. 

5.4.0.3.3 Sites of Application 

In addition to the types of efficacy claimed (e.g. bactericidal, fungicidal, tuberculocidal) and 
the  intended  area  of  use,  the  applicant  must  specify  the  use  patterns  for  which  the 
disinfectant is recommended on the label. 

Broad examples of use patterns (not exhaustive) could include areas such as: 

•  Use on intact skin; 

•  Use in hospitals, operating theatres, isolation wards, etc.; 

 
   
 
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•  Use in food manufacturing, retailing, processing areas etc.; 

•  Use in animal housing and equipment, e.g. pigs, sheep, poultry etc.; 

•  Use on work surfaces, cutting boards etc.; 

•  Use on fabrics or textiles; 

•  Use on toilets, bathrooms, sinks, etc.; 

•  Use against micro-organisms associated with human or animal waste; 

•  Use in air conditioning systems; 

•  Use in swimming pools, spas, aquariums and bathing waters; 

•  Use in tanks, pipelines, equipment soak or bottle wash. 

5.4.0.3.4  Directions for use (Methods of application) 

The label claim must specify the application method of the product. For disinfectants there is 
a  broad  range  of  application  methods  (e.g.  wiping,  aerosol,  spraying).  The  in-use 
concentration of the solution and the contact time, which are essential for safe and effective 
use, should be described on the label. Any other directions for use should also be specified, 
such  as  whether  the  surface  should  be  cleaned  first,  and  claims  regarding  the  number  of 
times  a  prepared  use  solution  can  be  used  (or  re-used)  before  a  fresh  solution  must  be 
prepared. 

The  application  method  can  have  a  strong  influence  on  the  efficacy  of  a  product,  therefore 
the  testing  of  a  product  should  be  appropriate  for  the  application  method.  If  specific 
equipment is used for application of the product (e.g. vaporisers) this should be taken into 
account  when  testing  the  product  for  efficacy.  Equipment  used  in  laboratory  tests  or  small 
scale tests may (of necessity) be different from that employed in practice. This is especially 
the case when biocidal active substances are generated in situ using large scale equipment, 
such  as  electrolysis.  In  cases  where  small  scale  tests  cannot  be  extrapolated  to  actual  use 
conditions a large scale test with the equipment should be done. 

5.4.0.3.5 Other interfering parameters 

Any other circumstances that can influence the efficacy of a product should be mentioned on 
the  label  (e.g.  temperature  or  pH  requirements).  For  example,  when  a  surface  should  be 
cleaned  before  applying  the  biocide  and  a  no  rinsing  step  is  involved,  or  that  alkaline 
cleaning fluids should not be used with acidic biocides, and vice versa. 

5.4.0.4  Efficacy testing 

For  efficacy  testing  of  disinfectants  in  general  only  quantitative  tests  methods  should  be 
used. 

5.4.0.4.1 Tiered approach 

For  efficacy  testing  of  disinfectants  a  tiered  approach  is  recommended.  The  following  tiers 
can be distinguished (in accordance with EN 14885): 

•  Phase  1  tests  are  quantitative  suspension  tests  to  establish  that  a  product  (or  an 
active  substance)  has  bactericidal,  fungicidal  etc.  activity  without  regard  to  specific 
conditions of intended use. Phase 1 tests cannot be used for any product claim. 

•  Phase 2 comprises two steps: 

o  Phase 2, step 1 tests are quantitative suspension tests to establish that a product 
has  bactericidal,  fungicidal,  virucidal  etc.  activity,  simulating  practical  conditions 
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o  Phase  2,  step  2  tests  are  quantitative  laboratory  tests,  often  using  carriers  or 
living  tissues  with  dried-on  micro-organisms,  simulating  practical  conditions  to 
establish that the product has bactericidal, fungicidal, virucidal etc. activity. 

•  Phase 3 tests are field tests under practical conditions. 

Phase 1 
Phase 1 tests are laboratory suspension tests to establish the basic activity of the product or 
active substance. These tests may be used during the development of the product, but are 
not accepted for product authorisation. However, a phase 1 test can be used to demonstrate 
that a co-formulant does not have any biocidal activity in the product. 

Phase 2, step 1 
Phase  2,  step  1  tests  are  laboratory  suspension  tests  in  which  the  ultimate  purpose  is  to 
establish  at  what  concentrations  the  product  meets  specified  requirements  under  “in-use” 
conditions.  In  these  tests,  in-use  conditions  (e.g.  temperature,  contact  time,  interfering 
substances) are considered in the test method. 

Various laboratory methods have been developed for biocide activity testing. Although these 
experiments differ in their design and experimental detail, they are all based on the principle 
of adding a test inoculum to the disinfectant (or vice versa) and taking samples at specified 
times.  The  biocide  in  each  sample  is  then  neutralised  and  the  survival  of  the  organisms 
assessed.  In practice, the methods can be  classified into two groups,  according to how the 
end-point of the test is determined: 

Quantitative tests 
Samples  of  untreated  and  biocide-treated  cells  are  plated  on  nutrient  medium  after 
neutralisation.  After  incubation,  the  number  of  colony  forming  units  is  determined  and  the 
log10 reduction in viable counts is determined. 

Capacity tests 
The biocide is challenged successively with the test organism at defined time intervals. This 
type  of test  can be used for instance for swimming pools  and toilet disinfectants which are 
challenged by new bacteria periodically. Following each inoculation, samples are taken, and 
after  a  suitable  contact  period  has  elapsed,  the  biocide  is  neutralised  and  the  sample 
incubated  in  a  suitable  growth  medium  to  determine  the  surviving  micro-organisms.  The 
result is expressed as the amount of the accumulated inoculum that was required to produce 
the “failure”. 

Phase 2, step 2 
Phase  2,  step  2  tests  are  simulated  use  or  practical  tests,  performed  under  rigorous 
conditions within the laboratory, which mimic real-life conditions, for instance by pre-drying 
the  micro-organisms  onto  surfaces.  These  tests  are  used  in  a  second  testing  stage.  After 
measuring  the  time-concentration  relationship  of  the  disinfectant  in  an  in-vitro  test  (phase 
2,  step  1),  these  practical  tests  are  performed  to  verify  that  the  proposed  use  dilution  is 
likely  to  be  adequate  in  real-life  conditions.  For  several  uses  standardised,  simulated  use 
tests exist (surface disinfection, hand wash or rub, instrument disinfection) but there are no 
standard tests available for many others. 

Longer-lasting  activity  is  claimed  for  some  products.  When  these  products  are  applied  to 
surfaces,  it  is  common  that  they  will  not  be  completely  removed  or  rinsed  off  after 
application. This might lead to longer-lasting activity of the biocide on the surface. Likewise, 
some  products  are  used  for  maintenance  via  continued  release  of  low  levels  of  biocidal 
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Phase 3 Field or in-use tests 
In-use testing involves the antimicrobial evaluation of the product under actual conditions of 
use on specified surfaces or materials in a specified environment. As with standard and non-
standard laboratory methods, representative organisms or actual organisms of concern may 
be used. 
Validated methodologies for these types of tests are currently not available, although some 
are in development. 

The practical use conditions under which a product can be used can be very variable and are 
therefore  difficult  to  standardise.  Field  tests,  although  not  standardised,  can  however  give 
valuable additional information on the efficacy of the product, provided that the studies are 
scientifically robust, well reported and provide a clear answer to the question. In these types 
of  test,  a  control  treatment  without  biocide  should  be  included.  Where  this  is  not  possible, 
efficacy should be judged on a comparison of the situation before and after application. 

Until validated standards are prepared, the responsibility for determining the acceptability of 
data derived from field trials in support of the claim will lie with the CA, taking into account 
the guidance given in EN 14885. 

5.4.0.4.2 Standard test methods 

Ideally,  data  should  be  generated  using  internationally  or  nationally  recognised  testing 
methods (CEN, OECD, ISO, etc.). Several international standard test methods currently exist 
for  disinfectant  products.  Recommended  standard  tests  are  presented  in  Appendices  2  and 
referenced in Appendix 4 to this guidance document. 

If  there  are  no  guidelines  available  for  the  specific  use  of  a  product,  or  guidelines  are  not 
suitable, the applicant  may use other methods (such as intra-company Standard Operating 
Procedures),  where  the  studies  are  scientifically  robust,  well  reported  and  provide  a  clear 
answer  to  the  question.  In  addition,  the  test  methods  used,  together  with  the  test 
conditions,  should  be  clearly  and  fully  described  and  must  address  the  efficacy  claim  that 
appears on the product label. The use of existing guidelines, with modifications to make the 
guideline more suitable for the specific product or use conditions, is also possible. EN 14485 
provides guidance on modification of standards (EN 14485, section 4.2 version 2014). 

At  the  time  of  publication  of  this  guidance  document,  a  broad  range  of  CEN  methods  are 
available. OECD has several phase 2/step 2 test methods developed for the efficacy testing 
of  disinfectants  to  be  used  on  hard  surfaces  which  have  been  published  as  Guidance 
Documents. Available tests are presented in Appendix 2 and referenced in Appendix 4. The 
use  of  CEN  test  methods  is  highly  recommended,  where  these  are  available  and  relevant. 
However  it  should  be  noted  that  although  this  Guidance  is  mainly  based  on  EN  standards, 
there are some cases where there are discrepancies compared to the EN tests. In such cases 
the ECHA Guidance should be followed as the leading guidance. OECD test methods may be 
used if, for example no CEN standard is available. 

These  methods,  described  below,  typically  give  a  standard  set  of  test  parameters,  test 
organisms  and  pass  criteria.  Where  specific  conditions  apply  for  a  field  of  use,  such  as 
high/low  level  soiling,  high/low  temperatures,  relevant  contact  times  etc.  these  conditions 
should be included in the efficacy tests. 

CEN Standard Test Methods 
A  Technical  Committee  (TC  216)  was  established  in  the  European  Committee  for 
Standardisation (CEN), to produce harmonised European methods for testing the activity of 
disinfectants  used  in  medical,  veterinary,  food,  industrial,  domestic  and  institutional  areas. 

 
 
 
 
 
   
 
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The  standards  are  based  on  suspension  tests  (phase  1  and  phase  2,  step  1)  and  some 
simulated use tests like surface tests (phase 2, step 2). 

European  standard  EN  14885  gives  information  on  the  application  and  interpretation  of 
European Standards for the testing of chemical disinfectants within product types 1, 2, 3 and 
4 of the Directive / Regulation. 

This  document  outlines  the  various  standards  currently  available  and  provides  guidance  as 
to  the  choice  of  available  standards  that  may  be  used  to  demonstrate  the  effectiveness  of 
disinfectants in particular situations (such as  medical,  veterinary and food hygiene) and  on 
the interpretation of results from such tests in making and supporting efficacy claims. 

In EN 14885 products intended for domestic use are grouped with products for use in food 
and  industrial  areas,  and  therefore  the  tests  specified  are  not  always  relevant  to  domestic 
areas.  For  instance,  the  virus  test  EN  13610  only  tests  against  bacteriophages.  In  these 
cases the test from the medical area should be used where relevant. In cases where no test 
method  is  available  for  one  area  of  use  (e.g.  sporicidal  test  in  medical  area),  a  test  from 
another area can be used instead, provided that the test parameters (soiling, temperature, 
etc.) are adapted to the intended use area (for further guidance on adaption of tests see EN 
14885 section 4.2). 

The  application  of  disinfectants  to  water  systems  such  as  swimming  pools,  spas,  and 
drinking water is not addressed in EN 14885. For the evaluation of activity against Legionella 
in aqueous systems (water used in cooling towers and water for general purposes, like spas, 
pools, showers and other uses) a quantitative suspension test is available (EN 13623). 

EN 14885 includes guidance on how a phase 3 field trial should be conducted. This guidance 
is intended to advise on the factors to be taken into account and controlled when performing 
a field trial. 

The  use  of  CEN  test  methods  is  highly  recommended,  provided  that  the  methods  are 
applicable  for  the  use  of  a  product.  In  some  cases,  the  method  can  be  adapted  (other 
contact times, soiling, etc.) to fit the use conditions. Any deviation from a standard must be 
clearly described and a justification for any deviations provided. 

OECD Standard Test Methods 
The OECD publishes practical test methods (comparable to phase 2, step 2 tests (1.4.1.3) or 
phase 3 (1.4.1.4)) for testing the efficacy of disinfectants on non-porous surfaces within the 
“Series on  Testing and Assessment”  or the “Series on Biocides”, respectively. Currently, all 
available  methods  have  been  issued  as  OECD  Guidance  Documents.  Guidance  Documents 
are,  however,  not  covered  by  the  Mutual  Acceptance  of  Data  (MAD)  principle  and  are 
advisory in nature. Further developed OECD  Test  Guidelines might become available in the 
future. As European Standards are not available for all types of applications yet, the use of 
OECD methods is  recommended provided that the methods are appropriately reflecting the 
use  of  a  product.  Again,  the  methods  can  be  adapted  (other  contact  time,  soiling,  etc.)  to 
better  fit  the  use  conditions,  provided  that  any  deviations  from  the  standard  are  clearly 
described and justified. 

Please  note  that  in  the  OECD  Guidance  Documents  on  disinfectants,  the  volume  of 
disinfectant solution added to the surface is very high compared to what is normally done in 
practice.  This  test  protocol  can  only  be  used  for  uses  where  the  volume  of  disinfectant 
solution per surface area is similar to the intended use (e.g. flooding). 

 
   
 
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Other Standard Test Methods 
While  CEN  standards  and,  in  case  no  CEN  standard  is  available,  OECD  methods  are  highly 
recommended, there are circumstances in which these tests cannot be applied, i.e. they are 
not  available,  or  relevant  to  a  particular  product  or  use  pattern.  In  those  cases  other  test 
methods can be used. 

Other test methods, for example VAH (former DGHM), DVG, AFNOR, US-EPA, AOAC or ASTM 
methods, are available and might be used when no international standard is available for a 
specific application. Where these methods lack predefined test parameters, target organisms 
or  pass  criteria,  the  applicant  has  to  provide  evidence  why  the  chosen  parameters  are 
appropriate for the intended application. 

Where  no  standard  tests  are  available,  suitable  test  protocols  may  be  designed  (and 
justified) by the applicant, but these should be discussed with and agreed by the CA before 
testing takes place. 

5.4.0.4.3 Data requirements 

Label claims and recommendations must be supported by the results of tests appropriate to 
the area of application. 

In  each  test  the  composition  of  the  product  to  be  tested  should  be  clearly  described, 
including the identity and function of the active substances specifying quality and quantity in 
the formulation. In addition, because the co-formulants can affect the efficacy if the product, 
they  must  also  be  clearly  described  including  identity  and  function.  Alternatively,  the 
formulation can be identified by a retrievable reference name or number. In such cases (i.e. 
it may only state a code for the product for the purposes of confidentiality), the composition 
of  the  tested  product  should  be  provided  separately.  As  the  formulation  may  affect  the 
efficacy  of  the  product,  the  composition  of  the  product  tested  should  be  the  same  as  the 
product under consideration. If not, justifications should be provided for any differences, and 
these will be assessed on a case-by-case basis. 

As phase 1 tests do not take practical use conditions into account, they are not considered 
acceptable  to  support  claims  during  product  authorisation.  In  general,  phase  1  tests  are 
used during the development of the product, for inclusion of active substances on the “Union 
list of approved substances” under the BPR or to prove that a co-formulant has no biocidal 
activity. 

In  general,  at  least  phase  2,  step  1  and  step  2  tests  are  required  to  support  label  claims 
during product authorisation. The phase 2, step 1 test will provide basic information on the 
efficacy  of  the  product  (in  a  standard  test),  while  phase  2,  step  2  tests  investigate  the 
effects  of  more  in-use  factors  (such  as  drying  of  target  organisms).  The  combination  of 
phase 2, step 1 and step 2 tests will generally provide a robust data package to demonstrate 
the efficacy of a product. Deviations from the tiered approach should be justified. 

In  some  cases,  for  example  when  disinfection  is  done  in  suspension  under  real  use 
conditions  (because  the  target  organisms  are  suspended  in  a  liquid  already  or  will  be 
suspended  during  the  process  due  to  mechanical  action,  for  example,  in  CIP),  a  phase  2, 
step 1 test is sufficient on its own, as this already simulates practical conditions.  

In other cases a phase 2, step 2 test may be replaced by a phase 3 test where a phase 2, 
step 2 tests is not appropriate. In general, a phase 3 test will be done in combination with a 
standard phase 2, step 1 test, as phase 3 tests are often variable. 

Where  in-use  conditions  cannot  be  simulated,  phase  3  tests  are  required  (e.g.  drinking 
water disinfection with ionisation equipment). 

 
 
 
 
 
   
 
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If more than one test method is available and applicable in phase 2, step 2 to substantiate a 
label claim for efficacy, it is sufficient to provide data from only one of the test methods. The 
test  method  selected  should  be  one  which  best  represents  the  way  in  which  the  product  is 
used.  For  example,  in  the  case  of  a  disinfectant  used  for  “hard,  non-porous  surfaces  by 
spraying”,  the  test  method  should  be  one  for  such  surfaces  without  mechanical  action  and 
with  representative  conditions  of  use,  such  as  contact  time,  soiling,  temperature  and  test 
organisms. 

It is not mandatory to perform the tests under obligatory test conditions of the standards if 
the  claimed  use  conditions  of  the  products  are  different  from  these  obligatory  tests 
conditions. 

Tests  have  to  be  performed  with  relevant  target  organisms,  which  are  selected  in 
accordance with the standard and the intended use of the product. This is further discussed 
in Section 1.3.1 of this Guidance. A list of standard test organisms is given in Appendix 3. 

The  concentrations  used  in  testing  should  be  selected  to  demonstrate  the  threshold  of 
product efficacy. Suspension tests should be performed with several dose rates, including at 
least one rate lower than the effective rate. Competent Authorities (CAs) will evaluate dose 
response  data  generated  in  these  tests  in  order  to  assess  if  the  recommended  dose  is 
appropriate  (i.e.  the  concentration  is  not  too  high,  or  at  the  minimum)  to  achieve  the 
desired effect. 

For  biocidal  products  which  claim  a  biostatic  effect  (bacteriostatic,  fungistatic,  etc.  i.e.  the 
ability  to  inhibit  growth  of  bacteria,  fungi  etc.  without  killing  them)  the  efficacy  should  be 
shown by suspension tests and simulated use tests (e.g. surface tests). The suspension test 
and  simulated  use  test  should  be  performed  with  and  without  neutralisation  and  with  a 
water  control  (where  water  is  tested  instead  of  the  product).  The  results  from  this  testing 
should show that the product prevents growth of the test micro-organism (i.e. a lower level 
of  test  organism  compared  to  the  water  control)  but  does  not  necessarily  inactivate  them 
(the micro-organisms survive in the test without neutralisation). 

Biocidal products that claim a biostatic effect bear the risk of development of organisms with 
temporary  or  permanent  reduced  susceptibility  (resistance).  For  this  reason,  efficacy  of 
these types of products has to be examined carefully. 

In case  of in  situ production of the active substance  or  when an apparatus is used to dose 
the active substance in the right amount to the water, the report should contain information 
on safety measurements concerning over and under dosing. 

Other products, which do not have biocidal or biostatic activity, might fall within the scope of 
the  BPR,  Article  3  1  (a)  “with  the  intention  of  destroying,  deterring,  rendering  harmless, 
preventing the action of, or otherwise exerting a controlling effect on, any harmful organism 
by  any  means  other  than  mere  physical  or  mechanical  action”  .  No  EU  standards  are 
available for these types of product yet, so applicants should provide a method following the 
principles  of  this  guidance  and  based  on  scientific  evidence.  During  development  of  new 
tests,  or  when  an  applicant  is  considering  using  a  non-standard  test  or  using  novel  testing 
methods, they should discuss this with the CA as to the acceptability and applicability of the 
test. 

In  the  following  sections,  guidance  on  the  requirements  per  product  type  and  use  will  be 
given. 

Detailed  but  non-exhaustive  lists  of  the  most  relevant  product  applications  and  uses  of 
biocides,  together  with  the  required  test  methodology,  are  given  in  the  claims  matrices 

 
   
 
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which  are  a  set  of  tables  linked  to  this  guidance  document  (see  Appendix  1  for  more 
information). 

For  uses  and  claims  that  are  not  specifically  mentioned  in  this  document  the  requirements 
will be set on a case by case basis by the CA. 

5.4.0.4.4  Relevant factors of the test procedure 

Formulation of the tested product 
A product authorisation is given to a single biocidal product with a defined composition or to 
a group of products making up a biocidal product family (BPF) and having  similar uses, the 
same  active  substances,  similar  composition  with  specified  variations  and  similar  levels  of 
risk and efficacy. 

With  respect  to  a  single  product  the  efficacy  of  its  specific  formulation  should  be 
demonstrated.  Therefore  it  is  important  that  the  formulation  tested  is  clearly  reported  in 
each  test  report  (or  provided  alongside  the  test  report  with  a  statement  that  it  is  the 
formulation  which  has  been  tested).  The  formulation  details  should  specify  the  active 
substances  and  co-formulants  present,  together  with  their  respective  concentrations,  and 
should  confirm  that  all  tested  formulations  contain  the  same  co-formulants  and 
concentrations.  Any  deviations  should  be  mentioned  and  justified  in  a  statement  or  in  the 
relevant  efficacy  reports.  Where  there  are  deviations  in  the  formulation  from  that  in  the 
product for which authorisation is sought, the tests will only be considered relevant where it 
is evident that the deviations have no effect on efficacy. In cases where this is not evident, a 
confirmatory study with the organisms that is most difficult to control should be proposed. 

Within  the  BPF  the  minimum  level  of  efficacy  over  the  whole  potential  range  of  products 
should  be  demonstrated  and  the  permitted  variations  in  composition  and  intended  uses 
should be explicitly identified. 

The  test  formulations  should  be  chosen  in  such  a  way  that  they  cover  the  whole  potential 
range  of  products.  The  test  formulations  should  include  at  least  a  product  with  the  lowest 
concentration  of  active  substance.  A  justification  should  be  given  whether  co-formulants 
influence  the  efficacy.  When  co-formulants  might  influence  the  efficacy,  the  worst  case 
concentration  of  co-formulants  (i.e.  low  concentration  of  a  co-formulant  that  might  have  a 
positive effect on efficacy, high concentration of a co-formulant that might have a negative 
effect on efficacy) should be tested. See also 1.5.7 for more information on testing BPF. 

Hard Water Claims 
The  degree  of  hardness  of  the  water  used  to  dilute  the  disinfectant  may  affect  its 
performance (by the presence of metal ions such as Ca2+ and Mg  2+). Generally the harder 
the  water  is,  the  less  effective  the  diluted  disinfectant  will  be.  Therefore,  test  programmes 
which  require  that  products  are  diluted  with  potable  water  must  be  diluted  in  water  of 
standard hardness as defined in the corresponding test standard, for the purpose of efficacy 
testing. 

It follows that any product that carries label claims for effectiveness in hard water must be 
tested by the appropriate method in water with defined hardness at the level claimed. 

Presence of Interfering Substances 
Where  disinfectants  are  applied  to  either  inanimate  surfaces  or  skin  or  liquids,  substances 
may be present on the surface or in the liquid, which may affect the disinfectant’s activity. 

The  nature,  amount  and  condition  of  the  soiling  present  will  affect  the  efficacy  of  a 
disinfectant. 

 
 
 
 
 
   
 
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In many cases residual contamination must be expected and in some situations (e.g. in the 
treatment of blood spillages) disinfectants are specifically used to decontaminate soiling, to 
prevent infection transfer and to assist in safe disposal. 

Blood,  urine,  faeces,  food  debris,  fats  and  oils,  dust  and  proteinaceous  materials  are  the 
most  likely  organic  soilings  to  be  encountered.  Limescale,  milkstone  and  soil  are  the  most 
common inorganic soilings. 

Where  claims  are  made  for  use  under  soiled  or  dirty  conditions,  the  use  concentrations  of 
the  product  must  be  determined  from  tests  performed  in  the  presence  of  suitable  soiling 
materials. Soiling materials commonly used in efficacy test methods include albumin serum, 
blood, yeast and yeast extract. 

In practice, with exception of a few situations (e.g. clean rooms), the presence of soiling on 
surfaces or in liquids to be disinfected cannot be ruled out. For this reason, a small amount 
of interfering substance should always be included during the testing of the product. In the 
CEN  methods  this  is  called  "under  clean  conditions".  Tests  under  clean  conditions  can  be 
used  when  the  surface  is  clean  before  disinfection.  This  is  for  instance  the  case  when  the 
label states that cleaning prior to disinfection is necessary. When a product claims combined 
cleaning and disinfection, the product should be tested under dirty conditions (see Appendix 
4 for more information). Also, where the label only states excessive dirt should  be remove, 
and the surface is still soiled after that (e.g. in the meat industry), soiling for dirty conditions 
should  be  used.  Please  note  that  in  some  cases  EN  14885  is  not  always  sufficient  to  meet 
BPR requirements. 

When a product is to be recommended for certain uses where the soiling is of a specific type 
(such as soap film residue or hard water scum), the product must be tested in the presence 
of  that  specific  soiling  type.  If  more  soiling  types  are  relevant  for  the  use  of  the  product 
(e.g. a product must be used in the beverage industry, in meat industry or in kitchens), pre-
testing should be done to determine the most challenging soiling type. Extended testing with 
the most challenging soiling type will be sufficient to cover all the others. 

As  an  exception  to  the  rule,  products  to  be  used  in  cleanrooms  do  not  require  additional 
soiling  in  the  test.  A  cleanroom  has  a  controlled  level  of  contamination  that  is  specified  by 
the  number  of  particles  per  cubic  meter  at  a  specified  particle  size.  The  soiling  level  in 
cleanrooms is  so low that even testing under  clean conditions  for the  EN tests is still over-
dosing  of  soiling  compared  to  cleanrooms.  For  these  uses  the  high  load  of  test  organisms 
can be seen as soiling. Tests without soiling will only be accepted when the label states the 
specific use in clean rooms which are classified according to ISO 14644-1 in class 1 to 9 or 
according to GMP EU classification in Grade A to D. 

Generally,  soiling  will  reduce  the  efficacy  of  the  disinfectant,  and  where  soiling  is  present, 
longer  contact  times,  higher  concentrations,  pre-cleaning  or  a  combination  of  these 
elements may be necessary. 

Temperature 
Generally,  disinfection  performance  increases  with  temperature,  although  this  depends  on 
the  active  substances  and  the  effect  on  individual  species  may  vary  depending  on  the 
specific  properties.  Therefore,  the  test  temperature  should  be  representative  of  those 
encountered  during  the  intended  use  of  the  product  (e.g.  low  temperature  in  animal 
housing, higher temperature in CIP). Some biocides are used in chemothermal disinfection, 
for instance, some CIP treatments are done under temperatures of 60-80ºC. Also for these 
uses the products should be tested at the use temperature. 

 
   
 
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If products (PT 2-4) are tested with high temperatures above 40ºC heat resistant reference 
test organisms must be used. Enterococcus faecium must be used as the only test organism 
for claiming bactericidal activity. For a virucidal claim the only test organism must be Murine 
Parvovirus. For a  sporicidal  claim the test  organism can be spores of,  for  example,  Bacillus 
cereus or Clostridium sporogenes. 

For  mycobacteria,  yeasts  and  fungal  spores  no  relevant  test  organisms  for  high 
temperatures  are  available.  Most  yeasts  and  fungal  spores  are  already  irreversibly 
inactivated by high temperature (>40 ºC) in the control without active substance. However, 
ascospores  of  several  fungi  can  become  heat  resistant  and  can  cause  problems  in,  for 
instance, the food industry. 

When  standard  tests  with  relevant  temperature  resistant  strains  become  available  for 
mycobacteria, yeasts and fungal spores, these should be used.  

When  efficacy  against  mycobacteria,  yeasts  and  fungal  spores  is  claimed  and  no 
temperature resistant strains are available, the standard test organisms should be tested at 
the maximum temperatures for which the test is validated.  

For  specific  claims  against  heat  resistant  species  (e.g.  Talaromyces  flavus)  efficacy  tests 
with these organisms should be provided. In these tests a control without biocide should be 
included which shows survival of the test organisms at the high test temperature. 

It  is  possible  that  the  concentration  needed  to  pass  the  test  is  higher  for  the  organisms 
tested  at  low  temperature  than  for  the  temperature  resistant  organisms  tested  at  higher 
temperature. In that case a justification should be given on how the test results reflect the 
use concentration in the use instruction on the label. 

Contact Time 
The contact time  of a product  on a  surface  etc. is an important aspect in the evaluation of 
the efficacy of disinfectants. In general, the longer the contact time, the more effective the 
disinfectant  is.  In  trials  where  test  organisms  are  taken  from  treated  samples  for  further 
analysis,  the  contact  time  between  the  biocide  and  the  test  organisms  should  be  stopped. 
Neutralisers,  membrane  filtration  or  subculture  techniques  are  used  to  prevent  residual 
carry-over of active substances. Neutralisation is discussed further in section 1.4.4.6 of this 
Guidance. 

Some  disinfectants  act  very  quickly,  whereas  others  require  an  extended  contact  time  to 
achieve  adequate  performance.  Mycobacteria,  bacterial  spores,  fungal  spores  and  non-
enveloped  viruses  take  longer  to  be  irreversibly  inactivated  than  most  vegetative  micro-
organisms. 

The  contact  time  that  is  practical  in  real  life  use  should  be  taken  into  consideration  when 
testing. In phase 2 and phase 3 tests the product should pass the test at the contact time 
recommended on the product label. 

Neutralisation 
Neutralisers  are  used  to  stop  the  product’s  activity  in  trials  where  the  test  organisms  are 
taken  from  treated  samples  for  further  analysis,  such  as  plate  count  following  biocidal 
treatment.  An  effective  neutraliser  for  the  test  product  should  be  identified,  and  evidence 
demonstrating  the  effectiveness  of  the  neutraliser  against  the  product  under  test,  and 
showing that the neutraliser itself does not have antimicrobial activity, must be included in a 
test report. Membrane filtration or subculture techniques can be used to stop the product’s 
activity, in combination with or instead of chemical neutralisation. These other methods are 
covered by the term “neutralisation” as used in this guidance. 

 
 
 
 
 
   
 
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Appropriate  controls  for  determining  the  efficacy  of  the  procedure  to  stop  the  product's 
activity after the contact time should be performed. 

pH 
The  prevailing  degree  of  acidity  or  alkalinity  during  disinfection  can  also  affect  the 
performance  and  choice  of  disinfectant.  Therefore,  the  pH  of  the  product  at  the  use 
concentration (diluted) as used in the test must be included in the test report. 

Texture of Surfaces 
Smooth impervious surfaces are easier to disinfect (and also to clean) than rough or pitted 
ones.  In  some  circumstances  the  micro-organisms  might  be  protected  from  the  action  of 
disinfectants by being protected in porous surfaces. Clumps of micro-organisms may also be 
more  difficult  to  inactivate,  as  cells  inside  are  protected  by  dead  micro-organisms  on  the 
outside.  Recently  porous  surface  tests  have  been  developed  (CEN)  to  test  under  these 
conditions. 

Bacteria  and  fungi  can  adhere  to  surfaces  forming  biofilms.  In  biofilms  susceptibility  is 
decreased (the bacteria are in a different physical state) and penetration  of biocide can be 
difficult  to  achieve  due  to  the  matrix  surrounding  the  bacteria.  This  makes  bacteria  in  a 
biofilm more difficult to inactivate. 

Repetition 
In  general  test  results  become  more  reliable  when  the  tests  are  done  in  replicates  (e.g. 
repeated  in  time,  in  more  test  objects).  Replicates  should  be  performed  as  required  in  the 
appropriate EN standards and where appropriate, internal standards or reference substances 
should be included. 

EN14885 section 5 (parts b, c and d) state the following information on precision of the test 
methods (repetitions): 

•  For standardised tests, or adaptation of a standard test, it is recommended to repeat 
the  test  and/or  include  an  internal  standard  and/or  performing  the  test  in  a  second 
and/or third laboratory. When doing the latter the second laboratory (and any further 
laboratory)  might  only  repeat  the  test  which  is  regarded  as  the  most  relevant  one 
with  the  least  susceptible  test  organism(s).  If  results  from  two  or  more  laboratories 
are  used,  each  laboratory  has  to  specify  one  result,  e.g.  “R  =  >  5.2  lg  (EN  13727-
instrument disinfection)”. Then the mean of the results of all laboratories is calculated 
assuming each laboratory’s result as equivalent. Results with lg “more than” are set 
as  this  figure,  e.g.  “>  5.2  lg”  is  used  for  calculation  as  “5.2  lg”.  All  lg  values  are 
converted to real numbers, e.g. 5,2 lg to about 158.000. The mean is the arithmetic 
mean of these converted numbers. If one of the testing laboratories obtains a result 
less  than  the  required  lg  reduction,  the  product  must  pass  if  further  tests  by  three 
other  laboratories  demonstrate  a  pass.  The  calculations  above  cannot  be  done  with 
tests where pass criteria are not expressed as lg reduction. 

• 

• 

In  case  of  repetition  of  the  test  it  is  unnecessary  to  repeat  the  test  with  all  test-
organisms but only with the least susceptible to the product under test. 

If two or more tests are carried out to support a claim of performance (e.g. phase 2, 
step 1 and phase 2, step 2) and the ensuing recommendation for use, the tests may 
be  ranked  according  to  their  order  of  relevance,  i.e.  their  ability  to  predict  the 
product’s performance under real life conditions. In case of a ranking only the result 
of the most relevant test may be repeated taking into account advice 3). If a ranking 
is  not  possible  only  the  results  of  the  test  showing  the  highest  minimum  active 
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5.4.0.5  General data requirements 

5.4.0.5.1 Test range 

Tests (phase 2, step 1) should be performed at a range of concentrations in order to verify 
that the use concentration is suitable for the desired  effect (e.g. not too high or not at the 
minimum effective level). 

5.4.0.5.2 Claim for several areas of use 

In cases where the product is intended for several areas, it is usually acceptable to perform 
the  tests  from  only  one  area,  as  long  as  the  test  is  performed  with  the  worst  case  test 
conditions  (temperature,  log  reduction,  interfering  substances,  etc.)  and  the  test  with  the 
highest/most  stringent  pass  criteria  is  used.  In  case  the  strains  are  different  between  the 
PTs all the strains must be tested.  

5.4.0.5.3 Biocidal products with biostatic effect 

For  biocidal  products  with  a  biostatic  effect  (bacteriostatic,  fungistatic,  etc.),  the  efficacy 
should  be  shown  by  suspension  tests  and  simulated  use  tests  (e.g.  surface  tests).  The 
suspension  test  and  simulated  use  tests  should  be  performed  with  and  without 
neutralisation. The results from these tests should show that the product prevents growth of 
the test  organism (no increase in numbers  compared to the negative control) but does not 
necessarily inactivate them (survival of the test organism in the test without neutralisation). 

5.4.0.5.4 Malodour control 

There  are  specific  requirements  for  products  claiming  control  of  organisms  that  cause 
malodour. Phase 2, step1 and step 2 tests should be performed with odour producing micro-
organisms.  A  justification  for  which  bacteria,  fungi,  etc.  are  relevant  to  the  intended  use 
should  be  provided.  Along  with  these  laboratory  tests,  an  odour  test  should  be  performed. 
The CA will decide on a case-by-case basis whether the product will receive authorisation. 

5.4.0.5.5 Changes in ingredients 10 

When  small  changes  are  made  to  the  non-active  ingredients  in  a  product,  it  is  not  always 
necessary  to  repeat  all  the  tests  with  the  new  formulation.  The  applicant  may  provide  a 
description of the changes and the effects that they have on the efficacy of the product. In 
the case of a minor change, a robust justification might be sufficient (to be decided by the 
CA). In other cases, new efficacy tests will have to be provided. This can be either a full set 
of efficacy tests or a test with the least susceptible organism in the former test. 

5.4.0.5.6 Treated articles 

See Section 5.3 for guidance on Treated Articles. 

5.4.0.5.7 Biocidal Product Families 

When  authorisation  is  requested  for  a  product  family,  efficacy  should  be  demonstrated  for 
the whole group but not necessarily of each product. More information is available in Section 
5.2 Product Families. 

5.4.0.6 Resistance 

See section 3.2 for guidance on resistance. 

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5.4.0.7 Assessment of application for authorisation 

5.4.0.7.1 Decision making 

Biocidal  Product  Regulation  528/2012  (Annex  VI)  stipulates  rules  for  decision  making  for 
biocides. 

The  test  results  must  meet  the  requirements  of  the  standards  or  other  criteria  for 
acceptance which are described below per type of use. Where a product does not conform to 
these  criteria,  the  applicant  should  provide  a  justification  in  the  application  as  to  why  the 
product should still be recommended for authorisation. The CA will decide on a case-by-case 
basis whether the product will receive authorisation. 

5.4.0.7.2 Assessment 

The  CA  assessor/expert  assesses  the  performance  of  the  product  as  demonstrated  in  the 
submitted efficacy tests against the label claims made for the product and the above criteria. 
If the product is judged to be sufficiently effective in laboratory (and, where relevant, field) 
tests, the product will be recommended for authorisation as far as efficacy is concerned. 

In  exceptional  cases  the  applicant  may  provide  justification  as  to  why  the  specified 
acceptance criteria are not met but the product is still acceptable. The CA will evaluate the 
justification on a case-by-case basis, possibly in consultation with the other CAs, and decide 
whether it is acceptable or not. 

The following sections give more specific dossier requirements per type of disinfectant. 

5.4.1 PT1 Human hygiene biocidal products 

5.4.1.1 Introduction 

Product type 1 contains biocidal products used for human hygiene purposes, applied on or in 
contact with human skin or scalps for the primary purpose of disinfecting the skin or scalp. 

Products  applied  on  human  skin  may  be  assigned  to  either  biocidal,  medicinal  or  cosmetic 
products or even to medical devices. If the product under investigation is within the scope of 
the  Medicinal  Products  Directive  (2001/83/EC),  the  Cosmetic  Products  Regulation  ((EC)  No 
1223/2009)  or  the  Medical  Devices  Directive  (93/42/EEC),  it  is  excluded  from  the  BPR  for 
the respective use. 

Products  for  disinfection  of  damaged  skin  (e.g.  wound  disinfection)  or  disinfection  of 
undamaged skin before a medical treatment of a patient (e.g. pre-operative skin disinfection 
before surgery and disinfection before injection) and products with a claim of medicinal use, 
are always medicinal products (covered by the Directive 2001/83/EC on medicinal products 
for human use). 

Biocidal products within PT1 are mainly hand disinfectants, which can include disinfection of 
wrist and forearm. 

When  applying  for  authorisation  for  a  biocidal  product  within  PT1  a  detailed  description  of 
the  intended  use  should  be  given,  to  prevent  authorisation  of  medicinal  products,  or 
cosmetic or medicinal uses, as biocides (e.g. the claim “skin disinfection” is insufficient). 

For  products  that  fall  under  the  BPR  the  data  requirements  described  in  the  following 
sections apply. 

 
   
 
 
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5.4.1.2 Hand disinfectants 

5.4.1.2.1 Introduction 

Hand  disinfectants  can  be  divided  in  hygienic  handwash,  hygienic  handrub,  surgical 
handwash and surgical handrub products. Handwash products are intended to be used with 
water,  handrub  products  are  intended  to  be  used  without  the  addition  of  water.  Hand 
disinfectants  can  include  disinfection  of  wrist  and  forearm.  Products  include  liquids,  gels, 
wipes, etc. 

Hand disinfectants can be used in a wide variety of areas such as hospitals and other health 
care institutions, food, beverage and other industry, private homes, etc. 

In  the  sections  below  the  requirements  and  acceptance  criteria  for  most  common  uses  are 
specified. For other uses and claims that are not specifically mentioned the requirements will 
be set on a case-by-case basis by the CAs. 

5.4.1.2.2 Data requirements 

Test methods 

For  efficacy  testing  of  hand  disinfectants,  the  tiered  approach  as  described  in  section 
5.4.0.4.1  of  this  Guidance  is  preferred.  For  hygienic  handwash,  hygienic  handrub,  surgical 
handwash and surgical handrub phase 2, step 1 and step 2 tests are required. Phase 2, step 
1  tests  are  available  for  all  relevant  test  organisms  and  required  depending  on  the  claim 
made.  For  a  claim  without  specification  of  the  area  of  use  the  phase  2,  step  1  for  medical 
area should be used. 

For bacteria a phase 2, step 2 test is available for these uses and therefore mandatory. For 
other  organisms  phase  2,  step  2  tests  will  be  mandatory  when  they  become  available.  For 
an overview of available EN tests see Appendices 2 and 4. 

Disinfectant towelettes/wipes  
For hand disinfectant wipes, phase 2, step 1 tests should be done preferably with the liquid 
extracted from the wipe or, if difficult to extract, use the liquid as it is before it is added to 
the  wipes.  Phase  2,  step  2  tests  for  hand  disinfection  (modified  EN  1500)  should  be  tests 
with the wipe applied on volunteers hands according to the intended use. The wipes should 
be used on full hands and not on the fingertips only. In addition, a test must be performed 
that shows that either the wipe will still disinfect if the wipe dries out or that the wipe stays 
wet  long  enough  to  disinfect  according  to  the  claim.  In  addition,  the  use  directions  can 
address these issues, for instance, stating on the label that only wet wipes are efficacious or 
giving  expiry  dates  for  re-sealable  packages  if  appropriate  according  to  the  intended  use 
conditions. 

Test organisms 

Hand  disinfectants  intended  for  general  hygiene  purposes  should  be  at  least  sufficiently 
effective  against bacteria and yeasts. Efficacy tests with these organisms should always be 
provided.  For  hand  disinfectants  intended  for  use  in  industrial  environments  to  prevent 
spoilage  of  products,  in  some  cases  also  prevention  of  bacteria  and  yeast  infections  is  of 
importance,  for  example,  in  food  and  cosmetic  industry.  In  other  industries  it  may  be 
justified that only efficacy against bacteria is sufficient. 

For all other groups of organisms tests have to be provided only when activity against those 
specific organisms is claimed. 

 
 
 
 
 
   
 
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The test organisms used in efficacy tests are normally stated in the applicable standard test 
methods.  

Additionally to the obligatory  species,  other species  can be used if there are  valid scientific 
arguments to justify their use, such as a need to show activity against specific organisms of 
concern  in  a  human  health  environment,  especially  emerging  health  risks,  or  in  specified 
industries. 

An overview of reference test organisms is given in Appendix 3. 

Virucidal activity 
For products used as hygienic hand disinfectants a differentiation in the virucidal activity is 
made.  

The claims can be: 

• 

• 

full virucidal activity; 

limited spectrum virucidal activity; 

•  activity against enveloped viruses. 

For each claim different test organisms should be tested. 

The  EN  1447611  test  for  virucidal  activity  gives  the  opportunity  to  either  test  for  full  or 
limited spectrum virucidal activity for hand disinfectants. For full virucidal activity Poliovirus, 
Adenovirus  and  Murine  Norovirus  should  be  tested.  Limited  spectrum  virucidal  activity  is  a 
claim  for  hygienic  handrub  and  hygienic  handwash  products  using  Adenovirus  and  Murine 
Norovirus  as  test  organisms,  thus  including  activity  against  the  test  viruses  and  all 
enveloped viruses (see Appendix 5 for a list of relevant viruses). Activity against enveloped 
viruses is currently being discussed to be included in EN 1447612 (test virus: MVA = Modified 
Vacciniavirus Ankara). 

When  only  the  limited  spectrum  virucidal  activity  or  activity  against  enveloped  viruses  is 
demonstrated  the  label  claim  cannot  be  “virucidal”.  The  SPC  should  clearly  state  which  of 
the possible virucidal claims was demonstrated. 

Non-professionals may not understand the difference between a virucidal claim and a limited 
spectrum  virucidal  claim.  Therefore,  the  instructions  for  non-professionals  should  be 
carefully worded. National hygiene agencies should decide how this can be communicated to 
the public and how the  label claim (in the SPC) should be phrased to  prevent misuse (e.g. 
limited  spectrum  products  will  not  be  efficacious  during  an  outbreak  of  Hepatitis  A  virus  or 
Enterovirus). 

Test conditions 

It is important that the tests are performed using the same contact time as claimed on the 
label. 

The contact time can be found in the relevant EN tests. In general the contact times are: 

• 

for hygienic handwash and handrub products the contact time is between 30 and 120 
seconds; 

11 The current published version is EN 14476: 2013 

12 At the time of publication of this Transitional Guidance, prEN 14476:2011 is under development and 
has been submitted to CEN. 

 
   
 
 
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• 

for hygienic handwash and handrub products used in medical area the contact time is 
usually 30 seconds for bactericidal, yeasticidal activity and activity against enveloped 
viruses; 

• 

for surgical hand disinfection products the contact time should not exceed 5 minutes. 

It  must  be  assured  that  the  disinfected  hands  stay  wet  during  treatment  (e.g.  by  applying 
enough  product  or  by  applying  the  product  several  times  if  the  volume  necessary  is  too 
much to apply at once). 

Phase 2, step 1 tests should be carried out with soiling (interfering substances) for  clean or 
dirty conditions depending on the intended use and according to the  relevant EN tests, i.e. 
EN  13727  and  EN  13624  (medical  and  veterinary  area)  or  EN  1276  and  EN  1650 13 
(industrial,  domestic,  institutional  area).  Dirty  conditions  in  phase  2,  step  1  tests  are 
mandatory  for  handwash  applications.  For  handrubs,  clean  conditions  in  phase  2,  step  1 
tests suffice if use instructions state that the product must be applied on visibly clean hands. 

For handwash products the phase 2, step 1 tests should be performed with a dilution of the 
product to take into account that the product is used on wetted hands. This is described for 
bacteria in test EN 13727 and a similar approach should be taken for other organisms. 

Phase  2,  step  2  tests  are  performed  without  additional  soiling  according  to  EN  1499,  EN 
1500  or  EN  12791.  The  soiling  needed  for  clean  and  dirty  conditions  can  be  found  in  the 
relevant  EN  phase  2,  step  1  tests  and  in  the  Table  of  Pass  Criteria  and  EN  Standards  (see 
Appendix  4  for  more  information).  Note  that  dirty  conditions  for  products  used  in  hospitals 
and health care differ from those in other areas of use. 

5.4.1.2.3 Acceptance criteria 

A product in PT1 will be assessed to be sufficiently effective if the required laboratory tests 
have been carried out (using the required test organisms and test conditions), and when the 
pass criteria for the tests have been met. 

Where pass criteria are available in the standard test, these should be met. For PT1 products 
the required log10 reductions are referenced in Appendix 4 or EN14885. 

Since the test methods for these types of products are generally established, deviations are 
not foreseen. If, however, deviations are considered necessary, they must be justified in the 
application.  The  CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the 
other CAs where appropriate, and decide whether it is acceptable or not. 

5.4.1.3 Other skin and scalp disinfection 

For  other  skin  and  scalp  disinfection  products  the  overlap  with  medicinal  and  cosmetic 
products  is  significant.  Only  products  that  are  not  covered  under  either  of  these  directives 
can be considered as PT1 disinfectants. 

5.4.1.3.1 Data requirements 

Test methods 

For other skin disinfection products the tiered approach as described in  section 5.4.0.4.1 of 
this Guidance is preferred: phase 2, step 1 and step 2 tests are required. 

The same phase 2, step 1 tests as required for hand disinfectants can be used. 

13 These tests will be adapted for hand disinfectants. 

 
 
 
 
 
   
 
 
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Currently,  there  are  no  European  phase  2,  step  2  standard  tests  available  for  other  skin 
disinfectants. However, test protocols may be designed by adapting existing standards (e.g. 
CEN methods involving volunteers) in a way that mirrors the respective application. 

Newly designed test protocols should be timely discussed with and agreed by the CA before 
tests are carried out. 

Deviations from the existing/future standards should always be mentioned and justified. 

For an overview of available tests see Appendices 2 and 4. 

Test organisms 

Disinfectants for other skin and scalp should be at least sufficiently effective against bacteria 
and yeasts. Efficacy tests with these organisms should always be provided. 

For phase 2, step 1 tests the standard organisms of these tests should be tested. 

For phase 2, step 2 tests either the standard organisms of these tests can be tested or the 
normal occurring microflora in volunteer tests. 

For all other groups of organisms tests only have to be provided when activity against those 
specific organisms is claimed. 

Justification for the used test organisms should be provided. 

The test organisms used in efficacy tests are normally stated in the applicable standard test 
methods. An overview of reference test organisms is given in Appendix 3. 

Test conditions 

It is important that the tests are performed using the same contact time as claimed on the 
label. The claimed contact time has to be a realistic value and should be justified for the use. 

Phase 2, step 1 and phase 2, step 2 carrier tests should be carried out with BSA as soiling 
(interfering  substances)  for  clean  or  dirty  conditions  depending  on  the  intended  use. 
Simulated-use studies with volunteers are in general performed without additional soiling.  

The soiling needed for clean and dirty conditions can be found in the relevant EN tests (see 
EN 14885, medical area) and referenced in Appendix 4. 

5.4.1.3.2 Acceptance criteria 

A  product  in  PT1  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated-use  tests  have  been  carried  out  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test, these should be met. For PT1 products 
the required log10 reductions are referenced in Appendix 4 or EN14885. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

 
   
 
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5.4.2 PT2 Disinfectants and algaecides not intended for direct application to 
humans or animals 

5.4.2.1 Introduction  

Product type 214 contains disinfectants and algaecides not intended for direct application to 
humans or animals. This includes inter alia: 

•  products  used  for  the  disinfection  of  surfaces,  materials,  equipment  and  furniture 

which are not used for direct contact with food or feeding stuffs; 

•  usage  areas  such  as  swimming  pools,  aquariums,  bathing  and  other  waters;  air-
conditioning systems; and walls and floors in private, public, and industrial areas and 
in other areas for professional activities; 

•  products  used  for  disinfection  of  air15,  water  not  used  for  human  or  animal 

consumption, chemical toilets, waste water, hospital waste and soil; 

•  products  used  as  algaecides  for  treatment  of  swimming  pools,  aquariums  and  other 

waters and for remedial treatment of construction materials; 

•  products used to be incorporated in textiles, tissues, masks, paints and other articles 
or  materials  with  the  purpose  of  producing  treated  articles  with  disinfecting 
properties. 

The data requirements  (test standards and test organisms) and assessment criteria for the 
most  common  uses  are  specified  below.  Detailed  but  non-exhaustive  lists  of  the  most 
relevant product applications and uses of disinfectants within PT2, together with the relevant 
test methodologies are given in the claims matrices which are a set of tables linked to this 
guidance document (see Appendix 1 for more information). 

All  of  the  possible  uses  in  this  PT  cannot  be  covered  in  the  matrices.  For  other,  less 
common, uses and claims that are not specifically mentioned, there is often no international 
standard tests available. Where this is the case, the applicant should provide tests that show 
the efficacy of the product and a justification for the use of these tests. The assessment of 
these products will be based on expert judgement and will be handled case-by-case. 

5.4.2.2 Data requirements 

There  are  some  general  data  requirements  that  apply  to  all  uses  in  PT2,  and  these  are 
described  below.  There  are  also  specific  data  requirements  that  apply  to  different  types  of 
use, and these are described in the sections covering those uses. 

The intended uses  of the disinfectant determine which tests will be  required to support the 
product.  Tests  that  most  closely  reproduce  the  practical  application  conditions  should  be 
selected. 

In  general  it  is  not  known  which  organisms  are  present  on  a  surface  or  matrix  to  be 
disinfected.  Therefore  a  disinfectant  must  have  a  broad  spectrum  of  activity,  in  order  to 
control all of the organisms that may be present. 

14 This includes biostatic products. 

15 This is taken to mean disinfection of air itself. Disinfectants sprayed or vaporised into the air (e.g. 
room disinfection by vaporised biocide) are generally for the purpose of disinfecting surfaces and not 
the air itself. Disinfectants for air conditioning systems disinfect the surfaces in these systems, not the 
air coming out of it. 

 
 
 
 
 
   
 
 
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5.4.2.2.1 Use in health care 

For  general  applications  in  the  medical  sector,  the  products  should  be  at  least  sufficiently 
effective  against  bacteria  and  yeasts  (which  are  responsible  for  most  common  nosocomial 
infections). Additionally, efficacy against other organisms can be claimed. 

Products intended to disinfect surfaces that are likely to come into contact with the patient 
and/or  the  medical  staff  and  surfaces  which  are  frequently  touched  by  different  people  a 
leading to the transmission of micro-organisms to the patient, must be tested with a contact 
time of maximum of 5 minutes. The same applies when the contact time of the product must 
be limited for practical reasons. Products for other surfaces than those stated above, may be 
tested with a contact time of a maximum of 60 minutes. 

5.4.2.2.2 Tuberculosis departments 

If the product is to be used in tuberculosis departments, the product should be efficacious as 
a  general  disinfectant  used  in  health  care  (efficacy  against  bacteria  and  yeast),  but 
tuberculocidal activity or mycobactericidal activity must also be demonstrated. 

5.4.2.2.3 Cleanrooms 

Products  to  be  used  in  cleanrooms  only  differ  in  the  data  requirements  for  the  interfering 
substance  to  be  used  in  the  tests.  As  an  exception  to  the  rule,  products  to  be  used  in 
cleanrooms do not require additional soiling in the test. A cleanroom has a controlled level of 
contamination  that  is  specified  by  the  number  of  particles  per  cubic  metre  at  a  specified 
particle  size.  The  soiling  level  in  cleanrooms  is  so  low  that  even  testing  under  clean 
conditions for the EN tests is still over-dosing of soiling compared to cleanrooms. For these 
uses the high load of test organisms can be seen as soiling. Tests without soiling will only be 
accepted when the label states the specific use in clean rooms which are classified according 
to ISO 14644-1 in class 1 to 8 or according to GMP EU classification in Grade A to C. 

5.4.2.2.4 Products against viruses 

Products  against  viruses  must  be  effective  against  viruses  with  and  without  an  “envelope” 
(protein  or  lipid  mantle).  Products  can  claim  virucidal  efficacy  if  efficacy  against  non-
enveloped  viruses  has  been  proven.  Such  products  can  be  regarded  as  efficacious  against 
enveloped and non-enveloped viruses. 

The  Phase  2  step  2  virus  test  described  in  EN  14476,  should  be  used  for  testing  products 
against  viruses  used  in  domestic  areas.  For  testing  products  used  in  veterinary  hospitals 
either EN 14476 or EN 14675 can be used. For products used in the medical area, a phase 2 
step 2 test is under development, see prEN 16777. 

5.4.2.3 Disinfectants for hard surfaces (in PT2) 

5.4.2.3.1 Introduction 

Biocides  can  be  used  to  disinfect  hard  surfaces  in  areas  such  as  hospitals  (including 
veterinary  hospitals,  dental  facilities  etc.),  industry,  institutions  or  private  homes.  These 
surfaces  may  be  tables,  floors,  walls,  the  outsides  of  machinery  and  hard  furniture,  etc. 
Products are often wiped or sprayed onto the surfaces and may be washed or wiped off after 
a certain contact time. 

The testing requirements for some  specific uses of hard surface disinfectants are discussed 
in  separate  sections,  for  example,  toilets,  room  disinfection  with  vaporised  biocide, 
immersion  of  equipment  into  the  product,  etc.  As  the  areas  of  use  can  be  as  diverse  as 

 
   
 
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private  homes  to  operating  theatres,  the  test  requirements  might  vary  depending  on  the 
area of use. 

5.4.2.3.2 Data requirements 

See  general  data  requirements  for  PT2  (see  sections  1.5  and  3.2  of  this  Guidance).  A 
detailed, but non-exhaustive list of the most relevant product applications and uses of hard 
surface  disinfectants  and  the  required  test  methodologies  are  given  in  Claims  Matrix  PT2, 
table  for  “Hard  surfaces”:  the  claims  matrices  are  a  set  of  tables  linked  to  this  guidance 
document (see Appendix 1 for more information and also Appendix 4). 

Test methods 

For efficacy testing of hard surface disinfectants, the tiered approach as described in section 
5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for a hard surface disinfectant: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a quantitative surface test (phase 2, step 2), 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, etc.). 

Tests  in  phase  3  are  optional,  as  no  validated  test  methods  are  available  yet.  Several 
methods for testing the efficacy of hard surface disinfectants are available. Appendices 2 and 
4 give a list of recommended test methods. 

The following documents are recommended for surface disinfection: 

• 

EN 14885: gives an overview of which EN phase 2, step 1 and step 2 tests to use 

for different uses, 

if CEN standards are not relevant or available for the use or organisms claimed the following 
documents are recommended if appropriately reflecting the application: 

• 

OECD  guidance  for  the  testing  of  chemicals:  Quantitative  method  for 
evaluating  activity  of  microbiocides  used  on  hard  non-porous  surfaces  (these  are 
surface tests which would be considered phase 2, step 2 tests), 

The  use  of  the  specified  tests  is  strongly  recommended  where  they  are  relevant  and 
appropriate.  Where  the  tests  are  not  appropriate  to  the  product,  other  tests  can  be  used, 
although a justification for the relevance of the tests used should also be provided. 

It is preferred that tests should be selected that correspond to the use area of the product 
(e.g.  tests  from  medical  areas  for  use  in  hospitals  and  tests  for  industrial  areas  for  use  in 
cosmetic industry). Where the product is intended for use in several areas it is acceptable to 
perform  the  tests  specified  for  only  one  of  the  areas,  as  long  as  the  test  with  the 
highest/most stringent pass criteria is used. In some cases where the  worst case cannot be 
clearly identified all areas must be tested. 

Currently  validated  surface  tests  with  and  without  mechanical  action  are  available  (EN  and 
OECD). Validated surface tests with mechanical action have been developed, and should be 
used for products that are intended to be used with mechanical action (EN 16615). 

Where  specific  conditions  apply  for  a  field  of  use,  such  as  high/low  level  soiling,  high/low 
temperatures,  relevant  contact  times  etc.  (see  section  5.4.0.4.4  of  this  Guidance),  these 
conditions should be included in the efficacy testing. 

 
 
 
 
 
   
 
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Disinfectant towelettes/wipes  
For  disinfectant  wipes,  the  phase  2,  step  1  tests  should  be  done  preferably  with  the  liquid 
extracted from the wipes, or if difficult to extract, use the liquid as it is before it is added to 
the  wipes.  Phase  2,  step  2  tests  should  be  tests  with  mechanical  action.  These  tests  are 
available for bacteria and yeasts. For testing other organisms surface tests can be done with 
liquid  extracted  from  the  wipes  (not  the  original  liquid),  with  a  justification  of  the  volume 
that is applied per square centimetre. In addition, a test must be performed that shows that 
either  the  wipe  will  still  disinfect  after  the  wipe  dries  out  or  that  the  wipe  stays  wet  long 
enough to disinfect according to the claim. In addition, the use directions can address these 
issues,  for  instance,  stating  on  the  label  that  only  wet  wipes  are  efficacious,  defining  the 
surface  area  each  wipe  can  disinfect  (e.g.  0.5  m2),  or  giving  expiry  dates  for  re-sealable 
packages. 

Test organisms 

The test organisms used in efficacy tests are normally stated in the applicable standard test 
methods.  For  use  in  veterinary  health  care  the  target  organisms  in  the  test  for  the 
veterinary area (PT3) should be used. 

If standard tests are not used (there will normally need to be a justification for this), the test 
organisms used to support a general claim should be demonstrated to be equivalent to the 
reference test organisms given in Appendix 3. 

Tests  with  test  organisms  other  than  those  mentioned  in  Appendix  3  are  acceptable,  if 
adequate scientific evidence is submitted on which the relevance of the test organism to the 
field of use can be judged. 

Also see the general data requirements PT2 for specific claims and minimum requirements in 
health care. 

5.4.2.3.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and,  where 
relevant,  field  tests  have  been  performed  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. 

If  the  test  does  not  provide  these  criteria,  the  general  criteria  referenced  in  Appendix  4  or 
EN 14885 can be taken as guidance for the level of reduction required. Deviations from the 
pass criteria are possible, but must be justified in the application. The CA will evaluate any 
justification on a case-by-case basis, consulting the other CAs where appropriate, and decide 
whether it is acceptable or not. 

5.4.2.4 Soft furnishings 

5.4.2.4.1 Introduction 

Disinfectants  for  use  on  soft  furnishings  are  intended  to  be  used  on  fabrics  in  the  home, 
institutional  environment,  healthcare  and  healthcare  facilities.  These  can  be  used  to  treat 
porous  soft  surfaces  such  as  curtains,  sofas,  upholstery,  mattresses  and  carpets.  The 
products are often sprayed onto the surfaces. 

5.4.2.4.2 Data requirements 

See general data requirements in sections 1.5 and 3.2 of this Guidance. A detailed, but non-
exhaustive  list  of  the  most  relevant  product  applications  and  uses  of  soft  furnishing 
disinfectants and the required test methodology is given in Claims Matrix PT2, table for “Soft 

 
   
 
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furnishings”: the claims matrices are a set  of tables linked to this guidance document (see 
Appendix 1 for more information). 

Test methods 

For efficacy testing of surface disinfectants for use on soft furnishing the tiered approach as 
described in section 5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for a surface disinfectant: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a quantitative surface test (phase 2, step 2); 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, etc.). 

Tests in phase 3 are optional as no validated test methods are available yet. 

Where possible, the phase 2,  step 1 test  should be  selected from EN 14885 from the table 
that  corresponds  to  the  use  area  of  the  product  (e.g.  test  from  medical  area  for  use  in 
hospitals and test for domestic areas for use in private homes).  

The  phase  2,  step  2  surface  carrier  test  can  be  derived  from  adaptation  of  CEN  TC  216 
surface  tests.  Instead  of  a  hard  surface  carrier,  carriers  could  be  made  of  suitable  fabric 
types.  ISO  20743  can  also  be  used,  or  other  quantitative  methods  including  textile  as 
carrier. EN 16616 is not relevant since this is done in washing machines. 

Test organisms 

The same test organisms as given for hard surfaces should be tested. See section 5.4.2.3.2 
test organisms, of this Guidance and Appendix 3. 

5.4.2.4.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and,  where 
relevant,  field  tests  have  been  performed  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard tests these should be met. 

If the test does not provide these criteria, the general criteria referenced in Appendix 4 can 
be  taken  as  guidance  for  the  level  of  reduction  required.  Deviations  from  the  pass  criteria 
are  possible,  but  must  be  justified  in  the  application.  The  CA  will  evaluate  any  justification 
on a case-by-case basis, consulting the other CAs where appropriate, and decide whether it 
is acceptable or not. 

5.4.2.5 Room disinfection with vaporised biocide 

5.4.2.5.1 Introduction 

Room disinfection involves the reduction and inactivation of micro-organisms on the surfaces 
of  the  walls,  floor  and  ceiling  of  the  room,  as  well  as  on  external  surfaces  of  the  furniture 
and equipment present in the treated room. The product is applied by airborne diffusion of 
an  aerosol,  a  smoke,  a  vapour  or  a  gas.  The  technical  characteristics  of  the  diffuser 
equipment play a central role, ensuring a homogeneous distribution of the biocide product in 
the  volume  of  the  room  and  reaching  all  surfaces  (including  ceilings  and  the  undersides  of 
horizontal  surfaces),  therefore  the  diffuser  equipment  contributes  in  a  decisive  way  to  the 
efficacy  of  the  product.  Manual  spraying  is  not  covered  in  this  section,  but  under  hard 
surface disinfection (see section 5.4.2.3 of this Guidance). 

 
 
 
 
 
   
 
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Room disinfection may not disinfect the inside parts of furniture, and will not disinfect the air 
itself, so these uses are not considered in this section. Room disinfection is therefore closely 
related  to  surface  disinfection  without  mechanical  action.  As  this  causes  complications  in 
cases of organic contamination, cleaning of surfaces is necessary prior to room disinfection. 

Process 
The application of the product consists of four phases: 

(1)   the preparation phase (required depending on type of active substance and application 
(relative  humidity, 

the  environmental  conditions 

procedure),  during  which 
temperature) are modified to an optimal level for the product; 

(2) 

(3) 

 the conditioning phase, during which the product is diffused into the room, in order to 
reach the effective concentration; 

 the  disinfection  phase,  which  corresponds  to  the  contact  time  required  to  obtain  the 
expected level of efficacy; 

(4)   the  terminal  phase,  which  includes  aeration  of  the  room  to  remove  any  disinfectant 
present in the air, or other procedures for inactivation of the active substance, before 
access of people or animals into the room can be permitted (see Figure 2 below). 

Figure 2: The various phases of a cycle of disinfection of an automatic process  

Concentration 
biocide product in the air 

of 

the 

Contact time 

Waiting 
reintroduction  of 
operator 

time  before 
the 

Time 
dispersal 

of 

3 

1 

2 

4 

Time (h) 

       Immobilization of the room to be disinfected 

Phases of the cycle: 

1: preconditioning (optional) 

2: diffusion  

3: phase of contact  

4: aeration 

Particular attention must be given to the dispersal time and contact time. The dispersal time 
is  the  time  necessary  to  reach  a  target  concentration  of  the  product  in  the  air  and  on  the 
surfaces to be disinfected in a given volume, while the contact time is the time necessary to 
reach the expected efficacy. 

 
   
 
 
 
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Note:  the  various  phases  of  the  cycle  presented  are  theoretical  and  can  be  adapted 
according to the process. The maintenance of a concentration of biocide in the atmosphere 
may be achieved by the regular introduction of additional biocide during the contact phase. 

5.4.2.5.2 Data requirements 

Test methods 

Airborne  disinfection  differs  from  direct  application  of  liquids  to  surfaces.  Therefore  the  EN 
phase  2,  step  2  standards  for  surface  disinfection  are  not  applicable  for  room  disinfection. 
The tiered approach is still possible, however, by using different test methods. 

The following tests are normally required for a room disinfectants:  

•  when applicable, a quantitative suspension test (phase 2, step 1);  

• 

semi-field  trial  such  as  European  standard  based  on  NF  T  72-281  (EN  standard  in 
preparation) for disinfection using airborne application (phase 2, step 2). 

The  CEN  phase  2,  step  1  tests  are  suitable  as  suspension  tests  under  clean  or  dirty 
conditions, although only applicable for products that can be tested in suspension (e.g. not 
for  gasses).  These  tests  are  not  sufficient  on  their  own,  and  should  be  combined  with  a 
semi-field trial for disinfection using airborne application. Where it is not possible to test the 
product in a suspension test, the semi-field trial will be sufficient. 

NF  T  72-281  was  developed  by  AFNOR  (the  French  standardisation  body)  in  1986,  and 
updated  in  2009.  This  standard  was  taken  as  a  start  to  develop  a  new  EN  standard  on 
airborne  disinfection  of  surfaces  (decision  taken  within  the  framework  of  CEN  TC  216  in 
November  2012).  This  semi-field  method  evaluates  the  efficacy  of  disinfectants  when 
vaporised  in  a  room  (automatic  diffusion  process)  or  when  sprayed  in  the  direction  of  a 
surface  (manual  application).  Only  application  by  vaporisation  is  discussed  in  this  section. 
Once this method has been finalised and adopted at European level, any method variations 
should be taken into account. 

Basic principles of room disinfection 
Inert  and  dry  carriers  infected  with  a  known  number  of  micro-organisms  (bacteria,  fungi, 
yeasts, mycobacteria, spores and viruses including bacteriophages) are placed in a room of 
defined  volume,  temperature  and  relative  humidity.  The  size  of  the  test  room  should  be 
relevant to the claims for the product. The carriers used are often stainless steel, but other 
relevant (generally non-porous) materials can also be used, such as glass or plastic. 

When the disinfection of textiles (curtains etc.) and other materials (e.g. wallpaper. filters in 
flow cabinets) is claimed, appropriate carriers should be used to demonstrate efficacy. 

The  inoculated  carriers  must  be  placed  in  a  vertical  position  with  inocula  facing  away  from 
the  diffuser.  Their  distance  to  the  diffuser  depends  on  the  room  dimensions  (for  instance: 
see  Appendix  B  of  NF  T  72-281).  The  test  method  defines  obligatory  test  conditions  for 
parameters that may influence the success of the disinfection. 

This test includes the use of milk in order to maintain viability of the micro-organisms on the 
carriers during the test. Depending on the area of use, suitable interfering substances should 
be tested (e.g. blood for use in hospitals). 

Similar  carriers  are  placed  in  a  second  room  nearby  which  is  not  treated  with  diffused 
product, to act as controls. 

Additional tests can be performed to simulate specific conditions that are encountered in the 
practice and to fit with label instructions. In this case, all experimental conditions should be 

 
 
 
 
 
   
 
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described clearly in the test reports. The standard lists the information that must be included 
in the final report. 

Diffuser 
As  mentioned  earlier,  the  disinfection  efficacy  is  closely  related  to  the  technical 
characteristics  of  the  diffuser.  Section  5  “intended  uses  and  efficacy”  of  the  Guidance  on 
information  requirements  for  biocides.  (Volume  II  Efficacy,  part  A)  requires  applicants  to 
take into account the technical equipment used, together with the product to be authorised. 

A detailed description of the equipment and its characteristics must be provided in sufficient 
detail to distinguish it from other equipment: 

•  equipment name and model; 

•  diffusion principles (e.g. fogging, vapour, fumigation) and particles size distribution of 

aerosols or powder; 

•  description  of  the  diffusion  performance  of  the  equipment  (e.g.  volume  to  disinfect, 

diffusion speed); 

•  description  of  the  ambient  conditions  (e.g.  humidity,  temperature)  in  which  the 

process can be used; 

•  diffusion time for a specific volume; 

•  precautions for over and under-dosing. 

The product authorisation will only be granted for use  with the equipment described in the 
application. After authorisation, any modification to the  equipment  should be validated and 
reported to the CA for evaluation.  

For major modifications that can affect the efficacy (e.g. pipe, pump, nozzles), it should be 
demonstrated that the efficacy of the process has not been affected (e.g. by a new study on 
the most resistant organism). 

For minor modifications that do not change the efficacy of the process, only a notification of 
the modifications to the equipment must be provided. 

Contact time 
As  room  disinfection  may  necessitate  a  long  period  of  treatment,  the  contact  time  to  be 
tested is not defined. The testing should demonstrate efficacy at a contact time proposed for 
the  intended  use.  This  should  be  relevant  to  practical  use  and  depends  on  substance 
concentration,  volume  of  room,  power  of  the  diffuser  equipment,  etc.  All  of  these 
parameters should be stated on the product label or in a technical information sheet. 

Test organisms 

Since  room  diffusion  is  used  to  disinfect  hard  (and  soft)  surfaces,  the  same  organisms 
should be tested as for hard surface disinfection (section 5.4.2.3 of this Guidance). Appendix 
3 contains a table of reference test organisms. 

The  general  data  requirements  for  PT2  for  specific  claims  and  minimal  requirements  in 
health care also apply for room disinfection with vaporised biocide. 

5.4.2.5.3 Acceptance criteria 

A product will be assessed to be sufficiently effective if the required laboratory and, (semi) 
field tests have been performed (using the required test organisms and test conditions), and 
when the pass criteria for the tests have been met. 

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If the test does not provide these criteria, the general criteria referenced in Appendix 4 can 
be  taken  as  guidance  for  the  level  of  reduction  required.  Deviations  from  the  pass  criteria 
are  possible,  but  must  be  justified  in  the  application.  The  CA  will  evaluate  any  justification 
on a case-by-case basis, consulting the other CAs where appropriate, and decide whether it 
is acceptable or not. 

5.4.2.5.4 Notes 

Limitations 

Any limitations of the procedure should be specified in the application. 

Literature  has  shown  that  disinfection  by  vaporised  biocide  may  not  be  as  effective  on  wet 
surfaces (lower concentration of the product) or inside closed cupboards and closets (where 
the  vapour  cannot  penetrate).  Therefore  carriers  should  be  tested  under  these  conditions, 
and  if  efficacy  is  not  proven,  the  label  instructions  should  provide  appropriate  information 
(such  as  stating  that  cupboard  doors  should  be  opened,  surfaces  should  be  dried  and  wet 
areas  (such  as  sinks  and  toilet  bowls)  should  be  disinfected  with  suitable  alternative 
products. 

Other factors which may influence the efficacy of the process in the practical use such as the 
equipment,  furniture,  special  structures  (e.g.  bumps  on  the  walls)  or  special  materials 
(copper  in  hydrogen  peroxide  procedures),  including  environmental  conditions  (e.g. 
temperature,  relative humidity) which may affect the  success  of the  disinfection, have also 
to be considered. The conditions of a sufficient vaporisation should also be specified. 

5.4.2.6 Swimming pools, spas and hot tubs 

5.4.2.6.1 Introduction 

Disinfectants are used to treat water in swimming pools,  spas and hot tubs. These may be 
public  pools  (which  may  be  used  by  many  people  daily)  or  household  pools  or  tubs  (which 
might  be  used  only  occasionally).  An  intermediate  situation  consists  of  facilities  in  hotels, 
housing complexes or sports clubs, where the bather load may be lower than in a fully public 
facility, but still high compared to private, domestic facilities. 

Disinfectant products can be added to a pool continuously, intermittently, by shock dosing or 
through  generation  in  situ.  Large  public  facilities  may  have  dedicated  staff  to  maintain  the 
pool using automated control systems, whereas smaller pools may be treated using manual 
methods  by  janitorial  staff.  Private  pools  may  be  treated  by  individual  householders, 
supplemented  in  some  cases  by  professional  pool  treatment  personnel.  Disinfection  is  only 
one aspect of pool maintenance and other activities, such as ensuring the correct pH and the 
removal  of  pollutants  by  oxidation,  flocculation  and  filtration,  are  essential  to  ensure 
adequate water quality. 

The principal purpose of the use of disinfectants is to treat the water to prevent the water-
borne transmission of pathogens between pool users. Supplementary purposes are to ensure 
the  aesthetic  quality  of  a  pool  (by  ensuring  that  algae  do  not  result  in  turbid  water  or 
unsightly and slippery microbial growth on pool surfaces, such as the floor and walls of the 
pool)  and  to  prevent  microbial  slime  and  biofilm  formation  in  pipework  and  related 
equipment. 

This  section  only  deals  with  disinfection  of  the  pool  water  and  the  pipework  and  related 
equipment  containing  pool  water.  The  disinfection  of  hard  surfaces  surrounding  the  pool  is 
covered in section 5.4.2.3 of this Guidance. 

 
 
 
 
 
   
 
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5.4.2.6.2 Data requirements 

See PT2 general data requirements in sections 1.5 and 3.2 of this Guidance. 

A  detailed,  non-exhaustive  list  of  the  most  relevant  applications  and  of  appropriate  test 
methodology is given in Claims Matrix PT2, table for “Swimming pools”: the claims matrices 
are a set of tables linked to this guidance document (see Appendix 1 for more information). 

Test methods 

For efficacy testing of pool disinfectants the tiered approach as described in section 5.4.0.4.1 
of this Guidance is preferred. 

The following tests are normally required for a pool disinfectant following a tiered approach:  

•  a quantitative suspension test (phase 2, step 1); 

• 

simulated-use tests with pool water or a surface test (phase 2, step 2)*;  

•  and a field test (phase 3)**; 

all simulating practical conditions appropriate to its intended use (temperature, contact time, 
soiling/bather load etc.).  

*   A phase 2, step 2 test may be appropriate in cases where a product has a specific use 
in  surface  disinfection.  Otherwise,  a  simulated  use  test  is  appropriate  for  products 
intended to disinfect the water in a pool or spa. 

**  In some cases the field trial can be waived. The OECD guidance document (described 
below)  is  based  on  experience  with  hypochlorous  acid/hypochlorite.  Therefore,  it  is 
acceptable that for products based on hypochlorous acid/ hypochlorite the field test is 
waived and only laboratory test data are provided. In some other cases, waiving the 
phase 3 test can also be justified. 

The OECD ”Guidance Document for Demonstrating Efficacy of Pool and Spa Disinfectants in 
Laboratory  and  Field  testing”  (OECD  Series  of  Testing  and  Assessment  No  170,  version 
dated 08 October 2012) describes laboratory and field test methods, conditions and criteria 
needed to demonstrate efficacy of a pool disinfectant. The protocol for field tests should be 
agreed between the applicant and CA before a field test is initiated. 

For  products  that  are  used  for  specific  purposes  such  as  disinfecting  pipework,  filters  and 
filter  media,  it  may  be  more  appropriate  to  test  using  the  EN  14885  methods  for  the 
disinfection of surfaces in institutional applications. 

Test organisms 

Besides bacteria and viruses, protozoa can also be of importance in swimming pools. Fungi 
may  pose  a  health  hazard  on  wet  surfaces  surrounding  the  pool  and  can  cause  slime  build 
up in pipework. OECD guidance lists the organisms that normally should be tested. Although 
algae and protozoa in pools are, in general only a problem when maintenance of the pool is 
not  carried  out  properly,  data  against  algae  and/or  protozoa  should  be  provided  where 
claims against these targets are made. 

5.4.2.6.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated use tests and where relevant, field tests have been performed (using the required 
test organisms and test conditions) and the pass criteria for the tests have been met. 

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The  OECD  guidance  document  sets  out  criteria  for  laboratory  and  field  tests.  However,  it 
may  be  noted  that  there  is  a  current  OECD  project  review  underway  to  look  at  criteria  for 
laboratory and field tests. 

Where  these  criteria  are  not  met,  the  applicant  can  provide  a  justification  as  to  why  the 
product  should  still  be  considered  acceptable.  However,  the  CA  will  evaluate  any 
justifications on a case-by-case basis, consulting the other CAs as necessary, and will decide 
whether it is acceptable or not. 

The OECD guidance document contains more details on factors to be considered. 

5.4.2.7 Toilets 

5.4.2.7.1 Introduction 

Biocides  can  be  used  to  disinfect  toilet  bowl  surfaces  in  diverse  environments  including, 
hospitals,  industry,  institutions  or  households.  Toilet  bowl  biocides  are  available  in  a  wide 
variety of forms, such as liquids, foams, powders, gels, pastes and tablets. 

These  products  are  often  applied  via  pouring  around  the  inside  rim  of  the  toilet  bowl 
surfaces with the area scrubbed after a minimum contact time. 

Other products are applied in the toilet permanently. They can be attached over the rim of 
the  toilet  bowl,  stuck  directly  onto  the  side  of  the  toilet  bowl,  placed  directly  in  the  cistern 
(water  reservoir),  or  attached  by  other  means.  These  products  are  normally  discharged 
when the toilet is flushed. 

Hard  surfaces  on  the  inside  of  toilets  are  covered  by  this  section.  Surfaces  on  the  outside 
and toilet seats, lids etc. are covered by section 5.4.2.3 “hard surfaces” of this Guidance. 

The  use  of  biocides  in  chemical  toilets,  most  commonly  found  on  airplanes,  trains,  and  in 
portable toilets, is not covered in this section, (see section 5.4.2.13 of this Guidance). 

5.4.2.7.2 Data requirements 

See PT2 general data requirements in 5.4.0.5 and 5.4.2.2. 

A  detailed,  non-exhaustive  list  of  the  most  relevant  applications  and  of  appropriate  test 
methodology is given in Claims Matrix PT2, table for “Toilet bowls”: the claims matrices are 
a set of tables linked to this guidance document (see Appendix 1 for more information). 

Test methods 

For  efficacy  testing  of  toilet  disinfectants  the  tiered  approach  as  described  in  section 
5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for a hard surface disinfectant: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a quantitative surface test (phase 2, step 2); 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
contact time, etc.). 

Several test methods for quantitative suspension and surface tests are available. 

Appendix  2  gives  a  list  of  recommended  test  methods.  The  following  documents  are 
recommended for surface disinfection: 

• 

EN 14885: gives an overview of what EN phase 2, step 1 and step 2 test to use for 

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if CEN standards are not relevant or available for the use or organisms claimed the following 
documents are recommended if appropriately reflecting the application: 

•  OECD guidance for the testing of biocides: Quantitative method for evaluating activity 
of  microbiocides  used  on  hard  non-porous  surfaces  (these  are  surface  tests  which 
would be considered phase 2, step 2 tests). 

The  use  of  the  specified  tests  is  strongly  recommended  where  they  are  relevant  and 
appropriate.  Where  tests  are  not  appropriate  to  the  product  other  tests  can  be  used, 
although a justification for the relevance of the tests used should also be provided. 

For products intended to be  added to the water reservoir  or hanging  down from the rim  of 
the  bowl,  the  concentration  of  the  product  (or  at  least  the  active  substance)  in  the  water 
before, between and after flushing should be determined. This can be done by an analysis of 
the  water  under  in-use  conditions  or,  for  products  where  all  parameters  are  defined,  by 
calculation. The laboratory efficacy tests should be performed using these concentrations. 

Tests in phase 3 are optional. 

When efficacy against biofilm is claimed a simulated-use test or field test has to be provided, 
next to a phase 2, step 1 test. See section 5.4.2.11 of this Guidance for test methods. 

Test organisms 

The same test organisms as for hard surfaces should be tested. See section 5.4.2.3.2 of this 
Guidance and Appendix 3. 

Products  will  normally  only  target  bacteria  and,  optionally,  yeasts  and  viruses.  Fungi  and 
spores  are  usually  not  relevant  in  the  toilet  bowl  but  when  efficacy  is  claimed  testing  is 
required. 

5.4.2.7.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and,  where 
relevant,  field  tests  have  been  performed  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard tests these should be met. 

If the test does not provide these criteria, the general criteria referenced in Appendix 4 can 
be  taken  as  guidance  for  the  level  of  reduction  required.  Deviations  from  the  pass  criteria 
are  possible,  but  must  be  justified  in  the  application.  The  CA  will  evaluate  any  justification 
on a case-by-case basis, consulting the other CAs where appropriate, and decide whether it 
is acceptable or not. 

5.4.2.8 Air-conditioning systems 

5.4.2.8.1 Introduction 

Disinfection of air-conditioning systems is similar to hard surface disinfection since only the 
surfaces  in  the  system  are  disinfected  and  not  the  air  itself.  The  difference  with  general 
surface disinfection is that the surfaces are mostly hidden inside the system and cannot be 
reached  easily  without  taking  it  apart  (for  instance  for  air-conditioning  systems  in  cars, 
dismantling the system would not be desirable). 

In general, disinfectants for air-conditioning systems are applied by airborne diffusion of an 
aerosol, a smoke, a vapour or a gas. The biocide is applied to an operating air-conditioning 
system  at  the  inlet  of  the  system.  This  way  the  biocide  is  sucked  into  and  passes  through 
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Preservation  of  cooling  liquids  is  not  covered  under  PT2  but  rather  within  PT11 
(preservatives for liquid cooling and processing systems). 

5.4.2.8.2 Data requirements 

For products that are applied by airborne diffusion of an aerosol, smoke, vapour or gas the 
same test methods and test organisms should  be used as  for  room disinfection.  Therefore, 
the same data requirements as for section 5.4.2.5 of this Guidance (Room disinfection with 
vaporised biocide) are applicable here. 

The following tests are normally required for a disinfectant for air-conditioning systems: 

•  when applicable, a quantitative suspension test (phase 2, step 1); 

• 

semi-field trial such as NF T 72-281 for disinfection using airborne application (phase 
2, step 2). 

See section 5.4.2.5 of this Guidance for specifications. 

In  the  semi-field  test  the  carriers  inoculated  with  test  organisms  are  placed  in  the  air-
conditioning system at the beginning and at the end of the system. When it is not possible 
to put carriers in the system they should be placed between the biocide application site and 
the  inlet  of  the  system  and  at  the  end  of  the  system,  in  the  out-flowing  air.  If  carriers  at 
both  sides  fulfil  the  criteria  it  can  be  assumed  that  the  surfaces  in  between  are  also 
disinfected sufficiently. 

For  products  that  are  applied  by  manual  spray,  the  test  methods  and  test  organisms  used 
for hard surface disinfection should be employed. See section 5.4.2.3 of this Guidance (Hard 
surface disinfection) for data requirements. 

In  addition  to  these  data,  the  applicant  should  provide  a  justification  that  the  spray 
apparatus is capable of reaching all (hidden) surfaces of the air conditioning system. 

5.4.2.8.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and,  where 
relevant,  field  tests  have  been  performed  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

The same pass criteria can be used as for other surface disinfection (section 5.4.2.3.3 of this 
Guidance). The criteria referenced in Appendix 4 can be used as guidance for what level of 
log10  reduction  is  normally  required.  Deviations  from  these  are  possible,  but  have  to  be 
justified in the application. 

5.4.2.9 Equipment disinfection by immersion 

5.4.2.9.1 Introduction 

Although  instrument  or  equipment  disinfection  can  be  considered  equal  to  hard  surface 
disinfection, it differs from the intended use in section 5.4.2.3 of this Guidance because it is 
mainly  applied  by  immersion  of  the  equipment  or  instruments  in  the  biocide  solution  or  by 
filling equipment with the solution (disinfection of inner surfaces). The products are intended 
for  equipment  used,  for  example,  in  health  care  facilities,  laboratories  and  industry.  The 
requirements  for  products  to  be  used  for  CIP  are  not  included  in  this  section  and  can  be 
found in section 5.4.4.3 of this Guidance. 

Some  of  the  products  used  for  disinfection  of  medical  instruments,  which  are  to  be  used 
specifically  for  diagnostic  and/or  therapeutic  purposes  for  human  beings,  do  not  fall  under 
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devices  or  a  group  of  medical  devices  (anaesthetic  equipment,  endoscopes,  surgical 
instruments,  incubators)  are  covered  under  the  Medical  Device  Directive  93/42/EEC. 
However, some disinfectants have a broader claim, for example, disinfection of instruments 
and surfaces. They are  so  called ‘dual use products’ as their distinct claims are covered by 
more  than  one  legislative  instrument.  The  BPR  states  that  such  biocidal  products  should 
comply,  in  addition  to  the  requirements  laid  down  in  the  BPR  with  the  relevant  essential 
requirements  set  out  in  Annex I  to  Council  Directive  90/385/EEC  of  20  June  1990  on  the 
approximation  of  the  laws  of  the  Member  States  relating  to  active  implantable  medical 
devices,  Council  Directive  93/42/EEC  of  14  June  1993  concerning  medical  devices  and 
Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in 
vitro diagnostic medical devices. 

5.4.2.9.2 Data requirements 

Test methods 

For  efficacy  testing  of  equipment  disinfectants  the  tiered  approach  as  described  in  section 
5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for an instrument disinfectant: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a quantitative carrier test (phase 2, step 2); 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, etc.). 

Methods for testing efficacy of equipment or instrument disinfectants are available. 

Appendix  2  gives  a  list  of  recommended  test  methods.  The  following  document  is 
recommended for instrument disinfection: 

•  EN 14885: gives an overview of which EN phase 2, step 1 and step 2 test to use for 

different uses. 

The  use  of  the  specified  tests  is  strongly  recommended  where  they  are  relevant  and 
appropriate. 

For  use  in  industry  and  institutional  areas,  no  specific  tests  for  instrument  disinfection  are 
given  in  EN14885.  Nevertheless,  phase  2,  step  1  suspension  tests  from  the  industry  and 
institutional areas can be used, by employing area specific soiling. For phase 2, step 2 tests, 
the  instrument  tests  for  medical  areas  are  most  appropriate.  Soiling  specific  to  the  area  of 
intended use should be employed. 

Test organisms 

For general disinfection of medical (including dental and veterinary) equipment, instruments, 
and  equipment  and  other  instruments  which  are  used  in  contact  with  skin  or  mucous 
membranes  (e.g.  instruments  for  pedicure),  efficacy  against  bacteria,  yeasts,  viruses  and 
fungal spores must be demonstrated. For instruments and equipment used in laboratory and 
industry the test organisms specified for hard surfaces should be tested. 

See section 5.4.2.3.2 of this Guidance and Appendix 3. 

5.4.2.9.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and,  where 
relevant,  field  tests  have  been  performed  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

 
   
 
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Where pass criteria are available in the standard tests these should be met. 

If the test does not provide these criteria, the general criteria referenced in Appendix 4 can 
be  taken  as  guidance  for  the  level  of  reduction  required.  Deviations  from  the  pass  criteria 
are  possible,  but  must  be  justified  in  the  application.  The  CA  will  evaluate  any  justification 
on a case-by-case basis, consulting the other CAs where appropriate, and decide whether it 
is acceptable or not. 

5.4.2.10 Textiles 

5.4.2.10.1 Introduction 

Biocides can be used to treat textiles and fabrics in hospitals, health care facilities, industry, 
institutions  or  private  homes,  when  relevant  micro-organisms  (pathogenic,  spoiling)  in  the 
textiles  have  to  be  reduced.  These  products  can  be  in  the  form  of  laundry  products  which 
combine  detergent  and  biocide  or  can  be  specialised  products  in  the  form  of  laundry 
additives which are  added to the wash  cycle or as finishing products (e.g.  fabric softeners) 
which are added in the last rinsing step or as pre-treatment. 

Typically contaminated clothes, linen or other washable textiles are treated in an appropriate 
washing machine. The biocide is added in concentrated form and diluted in the machine with 
water  according  to  the  specification  of  the  manufacturer  to  get  a  defined  concentration  in 
the  machine.  The  automated  chemical-thermal  process  normally  comprises  an  (optional) 
initial pre-treatment step for heavily soiled laundry, followed by the main washing step (at a 
defined temperature and defined contact time) and 3 to 4 rinsing steps with cold water. 

In some cases laundry can be treated through a hand-wash process in diluted biocide, which 
can take the form of a pre-soak (after which, machine washing is used), a hand wash only, 
or  through  soaking  to  disinfect  textiles  before  they  are  destroyed  (e.g.  in  an  infectious 
disease outbreak situation). 

Biocidal  laundry  products,  either  as  combined  biocide/detergent/conditioner  or  as  special 
additives,  are  available  for  either  targeted  pre-treatment  of  contaminated  articles  or  for 
whole-wash use. 

5.4.2.10.2 Data requirements 

See PT2 general data requirements in sections 1.5 and 3.2 of this Guidance. A detailed, non- 
exhaustive list of the most relevant applications and of appropriate test methodology  for is 
given  in  Claims  Matrix  PT2,  table  for  “Laundry  products”:  the  claims  matrices  are  a  set  of 
tables linked to this guidance document (see Appendix 1 for more information). 

Test methods 

For  efficacy  testing  of  textile  disinfectants  the  tiered  approach  as  described  in  section 
5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for a textile disinfectant: 

•  a quantitative suspension test (phase 2, step 1), 

•  a  quantitative  carrier  test  involving  carriers  made  of  test  fabric  (cotton,  polyester) 

(phase 2, step 2), 

Both  should  simulate  practical  conditions  relevant  to  its  intended  use  (concentration  of  the 
product,  temperature,  soiling,  different  fabrics,  contact  time,  etc.).  This  includes  the 
application of a normal washing procedure (including detergent) as a control. 

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•  phase 2, step 1 suspension tests as described in EN 14885, 

•  phase 2, step 2 tests involving 

o  a full-scale laundry machine test (EN 16616) 
o 

for  products  not  intended  to  be  used  in  washing  machines,  small  scale 
laboratory setting  (e.g. for pre-soaking in a bucket)  may be considered  (e.g. 
ASTM E4206 or ASTM E2274). 

In the phase 2, step 2 tests fabric is contaminated with test organisms and then exposed to 
the disinfectant. 

The EN tests are strongly recommended where available and appropriate. 

Test organisms 

Textile  disinfection  products  should  be  at  least  sufficiently  effective  against  bacteria  and 
yeasts. Efficacy tests with these organisms should always be provided. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. 

When disinfection is done at high temperatures (>40 ºC) relevant test organisms for these 
temperatures should be used as described in section 5.4.0.4.4 of this Guidance. 

An overview of reference test organisms, also for high temperatures, is given in Appendix 3. 

Test conditions 

For products intended to be added to washing machines, information on the following in-use 
conditions should be provided: 

• 

• 

• 

• 

the concentration of the product in the water during disinfecting process (i.e. washing 
or  rinsing).  The  water  volume  used  can  differ  between  wash  and  rinse  cycle  and 
different washing programmes, but also between washing machines; 

the water to textile ratio in the test is an important factor that should reflect the in-
use conditions; 

the  temperature  during  the  disinfection  process  (high  when  added  in  wash  process, 
low in rinse process); 

the  contact  time  (differs  between  various  washing  programmes  and  washing 
machines). 

The  laboratory  tests  should  be  performed  under  these  conditions.  The  conditions  for 
effective disinfection can normally only be carried out in professional washing machines. 

If  the  exact  conditions  cannot  be  met,  for  example,  in  household  machines,  reasonable 
worst case conditions must be tested. 

Worst case conditions, e.g.: 

• 

• 

• 

• 

the lowest temperature; 

the highest volume of water (i.e. maximum dilution of the product); 

the shortest contact time; 

the maximum load of laundry (i.e. smallest water to textile ratio). 

When  phase  2,  step  2  tests  involving  fabric  test  carriers  are  performed,  both  the  micro-
organisms  remaining  on  the  test  carriers,  those  released  into  the  washing  liquid  and  those 
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Manual soaking or pre-soaking can be done at room temperature but for some intended uses 
the temperature might start high and will cool down during the contact time (e.g. where hot 
water is used, which cools naturally). This should also be taken into account in the tests. 

Soiling 

The  interfering  substance  most  appropriate  for  the  in-use  conditions  should  be  used.  For 
instance,  blood  for  products  used  in  the  medical  area  and  protein  for  products  used  in 
industry,  institutional  and  domestic  areas  are  recommended.  The  soiling  on  a  domestic 
product for use in pre-soak (dirty clothes) will be very much higher than the soiling present 
for a post-wash rinse additive (clean clothes). For products used during pre-soak and wash, 
tests should be done under dirty conditions. For products used during post-wash rinse, tests 
should be done under clean conditions. 

5.4.2.10.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and,  where 
relevant,  field  tests  have  been  performed  (using  the  required  test  organisms  and  test 
conditions),  and  when  the  pass  criteria  for  the  tests  have  been  met.  When  the  product  is 
intended  to  be  used  in  combination  with  or  directly  after  a  detergent,  a  clear  effect  of  the 
disinfectant alone should be demonstrated. There should be a significant difference (+log 2) 
between disinfectant+ detergent and the detergent alone. 

EN and VAH tests provide pass criteria. 

No  acceptance  criteria  have  been  specified  in  the  ASTM  standards  for  laundry  (ASTM  E 
2406-04 or ASTM E 2274-09). 

If the test does not provide pass criteria, the general criteria  referenced in Appendix 4 can 
be  taken  as  guidance  for  the  level  of  reduction  required.  Deviations  from  the  pass  criteria 
are  possible,  but  must  be  justified  in  the  application.  The  CA  will  evaluate  any  justification 
on a case-by-case basis, consulting the other CAs where appropriate, and decide whether it 
is acceptable or not. 

5.4.2.11 Biofilms 

5.4.2.11.1 Introduction 

A biofilm is a complex aggregation of micro-organisms usually distinguished by the excretion 
of  a  protective  and  adhesive  matrix  attached  to  a  solid  surface  in  contact  with  a  fluid.  The 
matrix may incorporate other components derived from the environment. 

Once the first cell succeeds in attaching to a surface and a biofilm starts to form, growth of 
the biofilm may become very fast, as subsequent free floating bacteria find it much easier to 
attach to the developing matrix. 

Biofilms  can  grow  in  areas  such  as  inside  water  tanks  and  the  distribution  pipelines  of 
hospitals, hotels, industries and, in general, in any water systems which have temperatures 
and nutrients adequate for microbial growth. 

The  consequences  of  biofilm  formation  in  a  water  system  or  facility  may  be  severe 
depending on environmental conditions and any safety and performance requirements. 

In  healthcare  facilities,  biofilm  contamination  of  medical  equipment  or  water  systems  may 
increase the risk of nosocomial infections; in industrial facilities biofilm may cause microbial 
contamination  of  production  (pharmaceuticals,  cosmetics,  etc.);  in  other  situations  biofilms 
may  be  responsible  for  significant  reduction  of  the  performance  of  water  systems  by 
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Several  factors  may  contribute  to  biofilm  formation,  with  important  factors  including  the 
chemical composition and roughness characteristics of the pipe, tank or tube circuit. 

Bacteria  in  biofilms  are  more  resistant  to  disinfection  than  planktonic  bacteria  of  the  same 
species, as the presence of extracellular polymeric substances can act as a physical barrier 
to the biocide. This matrix may hamper biocidal penetration to the lower layers of the biofilm 
or may interact with the biocide and neutralise it. Additionally, the physiological state of the 
bacteria  in  the  biofilm  differs  from  bacteria  in  suspension,  which  can  also  influence  the 
susceptibility  of  the  bacteria  to  biocides.  Complex  communication  systems  are  often  also 
present that allow increased tolerance of members of the biofilm community to be initiated. 

Two types of activities of biocides against biofilm can be identified: 

1)  Prevention  of  biofilm  formation:  the  biocide  acts  on  biofilm  formation  (i.e.  in  this 
case  the  biocide  is  present  before  the  biofilm  is  formed  and  may  affect  the  early 
adhesion of cells to the surface or the viability of the cells); 

2)  Biofilm disinfection (“curative”): the biocide acts on a mature biofilm (i.e. when the 
biofilm  is  already  present  on  a  surface  and  the  biocide  interacts  with  the  biofilm-
embedded cells, with a -cidal effect). Biocidal products of this type may also achieve 
detachment of the biofilm (possibly in conjunction with physical action). 

In case where the biofilm is not  removed as a  result of the biocide treatment, it should be 
followed by mechanical removal of the biofilm. 

Industry  is  increasingly  developing  new  technologies  for  prevention,  inactivation  and/or 
detachment  of  biofilms  and/or  inactivation  of  biofilm  embedded  organisms,  for  example 
through  the  use  of  UV  light,  water  ionization  or  impregnated  or  coated  materials  and  new 
biocides which claim specific efficacy against biofilms. 

5.4.2.11.2 Data requirements 

There  are  currently  no  standard  laboratory  tests  available  to  verify  the  efficacy  of  biocides 
against biofilms. As this is an area in which the science is developing rapidly, the information 
below  should  be  considered  as  general  guidance  reflecting  the  state  of  knowledge  at  the 
time of writing this Guidance. 

Tests  to  demonstrate  the  efficacy  of  disinfectants  according  to  EN  and  OECD  are  based  on 
simpler  models  than  are  found  in  biofilms.  The  available  surface/carrier  tests  are  not 
representative  of  biofilm  models,  as  they  do  not  consider  the  presence  of  extra  cellular 
polymeric substances which act as a physical barrier to the biocide. 

Other  characteristics  of  the  biofilm  and  biocidal  product  should  be  taken  into  account.  For 
example, if biocide impregnated materials claim a preventive effect on biofilm formation, the 
prevention  of biofilm formation should be demonstrated, taking into  consideration the half-
life  of  the  impregnating  substance  which  may  differ  depending  on  the  material 
characteristics.  The  active  substance  may  be  released  from  the  surface  and/or  may  be 
inactivated by environmental factors. 

A standard suspension test can only be used to confirm basic activity of the product against 
the claimed organisms in a tiered approach. 

A suggested general approach could be: 

1)  a suspension test: any biocide claiming to act on biofilm, has to be first evaluated in 

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2)  a  simulated  use  efficacy  test  to  demonstrate  the  ability  of  the  product  to  exert  a 
controlling effect on the biofilm under either static condition or under flow conditions 
depending on the use pattern (claim). This controlling effect can be to destroy and 
detach, inhibit or prevent the formation of a biofilm; 

3)  a field trial, where the biofilm is formed under (simulated) use conditions. 

These  tests  should  be  performed  in  sequence  to  obtain  more  complete  information  on  the 
activity of the product on biofilm. 

For  biofilm  disinfection  (curative)  a  suspension  test  (as  for  (1)  above)  and  suitable  robust 
data from either a simulated use test (2) or field trial (3) should be performed. If there are 
no robust data from a simulated use test (2), a field test (3) is mandatory. 

For biofilm prevention the approach is different to that for biofilm disinfection, as the biocide 
is  present  before  the  biofilm  is  formed  and  may  affect  the  early  adhesion  of  cells  to  the 
surface  or  the  viability  of  the  cells.  In  this  case  the  suspension  test  (1)  may  not  be  useful 
since the product might not have a –cidal effect. 

Test methods 

Suspension tests 
The first step in the tiered approach is a suspension test. The CEN phase 2, step 1 tests are 
suitable  as  suspension  tests.  This  test  is  only  applicable  for  products  that  can  be  tested  in 
suspension and which have a –cidal effect. 

Simulated use tests 
Standard laboratory tests to verify the efficacy of biocides against biofilms are not currently 
available.  Therefore,  before  performing  a  biofilm  test,  the  methods  should  be  agreed  with 
the CA. 

Applicants  should  provide  a  method  following  the  principles  in  this  guidance  and  based  on 
scientific  evidence.  During  development  of  the  tests  CAs  of  member  states  should  be 
consulted to make sure that the tests are acceptable. 

Biofilms  can  be  formed  and  evaluated  in  static  or  flow  conditions.  The  way  the  biofilm  is 
formed  has  an  effect  on  the  susceptibility  of  the  biofilm  to  biocides:  biofilms  formed  under 
flow  conditions  are  generally  more  resistant  to  biocides  than  biofilms  formed  under  static 
conditions. 

The  conditions under which the biocidal products will have to  operate  should also be taken 
into account. Under static conditions the disinfectant operates without the aid of the removal 
effect of a fluid flow or shear stress. Under flow conditions the contact time might be shorter 
when shock dosing is used. 

Static tests are less expensive and easier to standardise, but flow tests are generally closer 
to the real use scenarios. 

In  both  cases,  the  reproducibility  and  repeatability  of  results  over  time  should  be  ensured; 
so a method that allows a series of observations, rather than a single observation, should be 
employed. 

Laboratory  tests  for  evaluating  the  efficacy  of  biofilm  disinfectants  should  emulate  the 
critical  factors  of  a  real-world  environment.  In  most  instances,  a  biofilm  will  not  be 
comprised of a single species and tests based on consortia relevant to the end use should be 
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In cases where only efficacy against biofilms formed under static conditions is claimed (e.g. 
use in tanks without flow) it is sufficient to only test against these biofilms. 

Examples  of  methods  for  testing  under  flow  and  static  conditions  are  described  below,  but 
other protocols are available in literature or may be under development. 

Static condition assay 
Standard  laboratory  tests  to  verify  the  efficacy  of  biocides  against  biofilms  formed  under 
static  conditions  are  not  currently  available.  However,  literature  describes  several  methods 
of how to create a biofilm in the laboratory under static conditions. 

An  example  of  a  semi-quantitative  method  for  biofilm  evaluation  is  the  microplate  test, 
where a biofilm is formed in static conditions and the amount of biofilm can be quantified by 
spectrophotometric measurements. The amount of living cells in the biofilm before and after 
treatment can also be determined. In this case, the disinfectant operates without the aid of 
the removal effect of a fluid flow or shear stress. 

A positive aspect of such an assay is that it is a low cost, easy-to-conduct test, that allows 
several  replicates  and/or  the  testing  of  several  conditions  (several  biocide  concentrations, 
more species, etc.) to be performed, which would provide the basis for a more accurate and 
closer-to-reality test. 

This method consists of the formation of a biofilm by the species of interest on the bottom of 
96 well plates (the material and coating of the plates should be specified); the disinfectant 
may  be  present  before  (preventive  effect)  or  after  (inhibition/removal  effect)  the  biofilm  is 
formed. The amount of biofilm (biomass) is quantified after staining of the adherent material 
and  spectrophotometric  measurement.  Detecting  chemicals  such  as  ATP  to  measure 
bacterial viability may also be used. 

Flow condition assay 
Standard laboratory tests to verify the efficacy of biocides against biofilm formed under flow 
conditions are not currently available. However, systems to generate a standard biofilm have 
been developed by CEN (CEN ISO/TS 15883-5:2005 Annex F) and ASTM (ASTM E2196 and 
ASTM  E2562).  Using  either  of  these  reproducible  biofilms,  a  method  for  the  assessment  of 
prevention  and/or  elimination  of  biofilm  in  terms  of  viable  cells  reduction  and  bacterial 
biomass reduction can be carried out. 

The  CEN  method  consists  of  the  production  of  a  standard  Pseudomonas  aeruginosa  biofilm 
inside a Teflon tube, using a flowing system to simulate a real world situation. 

ASTM  E2196  and  ASTM  E2562  standards  use  biofilm  rotating  disc  reactors,  which  are 
especially suited for high shear forces. 

The biofilm is then treated with a disinfectant to evaluate the biocidal capacity to remove or 
to reduce the biofilm. 

Other carrier types (e.g. silicon, steel, PVC, etc.) can be selected and used depending on the 
biofilm  development  system,  and  the  experimental  conditions  can  be  adapted  to  compare 
the efficacy of different treatments in preventing biofilm formation. 

A  reference  substance  of  known  activity  must  be  tested  in  parallel  (e.g.  chlorine  dioxide, 
sodium hypochlorite). 

Field trials 
As  for  other  situations  in  which  biocides  are  used,  only  field  tests  (phase  3  tests)  are  fully 
representative  of  the  activity  of  the  biocide  on  biofilms,  but  these  tests  are  difficult  to 

 
   
 
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standardise, and such tests should be complemented by laboratory suspension or simulated 
use tests, which have a higher degree of robustness and reproducibility. 

A  field  trial  should  reproduce  the  in-use  conditions  of  the  worst-case  situation  of  the 
intended uses. 

Prevention  and/or  elimination  of  biofilm  (in  terms  of  viable  cells  reduction  and  bacterial 
biomass reduction) should be demonstrated by sampling before and after disinfection. 

A field test can be waived if a suitably robust simulated use test, which adequately mimics 
the in-use conditions is provided. A robust test could for instance be a complex pipe system, 
in  which  natural  biofilm  formation  takes  place,  either  in  combination  with  the  addition  of 
standard organisms or not. 

Test organisms 

The choice of micro-organisms for a test is relevant, since the use of only one organism per 
test  is  limitating  and  may  not  be  fully  representative  of  the  real  events  leading  to  micro-
organism aggregation (biofilms in settings where disinfectants are used, are normally multi-
microbial,  i.e.  composed  of  several  different  species).  Moreover,  contaminants  from 
environmental  sources  may  be  embedded  in  the  biofilm  matrix  which  may  reduce  the 
disinfectant’s efficacy. 

Bacteria  are  not  the  only  inhabitants  of  biofilms,  as  both  fungi  and  algae  may  also  inhabit 
biofilms.  Protozoans  that  consume  bacteria  may  feed  on  biofilms.  Protozoan  oocysts  and 
virus  particles  can  become  entrapped  in  a  biofilm  and  later  detach,  returning  to  the 
environment. 

In  a  suspension  test,  the  standard  organisms  per  claimed  group  (bacteria,  fungi,  etc.) 
should be tested.  

For a general claim of efficacy against biofilms, as a minimum, bacteria should be tested in 
laboratory  biofilm  tests.  When  action  against  other  groups  of  organisms  (e.g.  fungi,  algae 
etc.) is claimed these should be tested as well. 

In suspension tests the standard organisms should be tested (see Appendix 3). 

Pseudomonas  aeruginosa,  Staphylococcus  aureus  and  Legionella  spp.  are  acceptable  test 
organisms for the laboratory biofilm tests. Mixtures of test organisms for producing biofilms 
are only acceptable as additional tests as it is difficult to standardise such methods. 

In  simulated  use  or  field  trials  the  biofilm  may  be  formed  in  vivo  with  naturally  occurring 
micro-organisms. 

5.4.2.11.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and,  where 
relevant,  field  tests  have  been  performed  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard tests these should be met. 

If the test does not provide these criteria, the general criteria referenced in Appendix 4 can 
be  taken  as  guidance  for  the  level  of  reduction  required.  Deviations  from  the  pass  criteria 
are  possible,  but  must  be  justified  in  the  application.  The  CA  will  evaluate  any  justification 
on a case-by-case basis, consulting the other CAs where appropriate, and decide whether it 
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5.4.2.12 Soil 

Disinfection of soil and other substrates (in playgrounds) with biocides is not common (and 
so far not claimed for Annex I of the BPD or the “Union list of approved substances” of the 
BPR). This is more often done for plant protection. Therefore, plant protection guidelines and 
EPPO  standards  on  soil  treatments  should  be  referred  to  for  test  methods.  The  use  of  the 
test methods should be justified with the application. 

5.4.2.13 Other uses in PT2 

Several other uses are mentioned in the description of PT2: wastewater and hospital waste 
disinfection,  algaecides  for  swimming  pools  and  indoor/outdoor  aquatic  area  (aquaria  / 
garden  ponds),  foot  baths  in  swimming  pools,  chemical  toilets,  disinfection  of  air.  No  data 
requirements and acceptance criteria for these uses are currently available. 

However, the general principles for efficacy evaluation in PT2 are  applicable for these other 
uses.  Efficacy  data  should  be  adequate  to  demonstrate  efficacy  and  suitability  for  the 
intended use, based on laboratory and/or practical data from existing and/or proposed new 
quantitative  studies.  If  desired  the  design  of  any  proposed  efficacy  tests  may  be  agreed 
between  the  applicant  and  the  CA  taking  into  account  all  relevant  conditions  of  use.  Such 
factors include consideration of the organisms to be controlled, requirements for biocidal or 
biostatic  effects,  contact  time  and  temperature  and  the  nature  and  presence  of  interfering 
substances. 

Specific  requirements should also be set  on  a  case by  case basis by the CA as appropriate 
for specific claims. 

5.4.3 PT3 Veterinary hygiene biocidal products 

5.4.3.1 Introduction 

Product  type  3  contains  biocidal  products  used  for  veterinary  hygiene  purposes  such  as 
disinfectants,  disinfecting  soaps,  oral  or  corporal  hygiene  products  or  with  anti-microbial 
function.  Products  used  to  disinfect  the  materials  and  surfaces  associated  with  the  housing 
or transportation of animals are also included. 

Some  of  the  products  in  PT3  are  on  the  borderline  with  veterinary  medicinal  products  or 
cosmetic  products.  If  the  product  under  investigation  is  within  the  scope  of  the  Veterinary 
Medicinal  Products  Directive  (2001/82/EC  as  amended  by  2004/28/EC)  it  is  excluded  from 
the  BPR  for  the  respective  use.  When  a  product  only  has  a  cosmetic  claim  (e.g.  cleaning 
skin,  hoofs,  paws)  and  no  reference  is  made  to  any  biocidal  claim  (e.g.  skin  disinfection, 
activity against micro-organisms), it is excluded from the BPR. 

Borderline cases are discussed in more detail in the respective sections below. 

Following a decision taken at the CA meeting in May 2015 (CA-May -2015-Doc 8.3) products 
applied for general disinfection of surfaces in the medical area (medical practices, hospitals) 
as  well  as  of  surfaces 
in  veterinary  practices  associated  with  examination  and 
operation/treatment  of  the  animals  are  assigned  to  PT  2,  whereas  products  for  specific 
veterinary  hygiene  purposes  (e.g.  products  with  specific  claims  against  a  target  organism 
only relevant in the veterinary area) are considered to be in PT 3. 

In  the  sections  below  the  requirements  and  acceptance  criteria  for  most  common  uses  are 
specified. For other uses and claims that are not specifically mentioned the requirements will 
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5.4.3.2 Disinfectants for hard surfaces in PT3 

5.4.3.2.1 Introduction 

Biocides can be used to disinfect hard surfaces, both porous and non-porous, in areas such 
as  animal  housing  (stables,  cages,  housing  for  pets,  etc.),  animal  transportation  vehicles 
(including tyres), hatcheries, etc. These surfaces may be tables, floors, walls, the outsides of 
(milking) machinery (including milking robots and milking clusters/claws) and hard furniture, 
equipment,  boots,  etc.  Products  may  be  applied  by  spraying,  wiping,  foaming  or  soaking, 
and  may  be  washed  or  wiped  off  after  a  certain  contact  time.  Boots  and  tyres  may  be 
disinfected  by  walk-through,  drive-through  bath  or  mat,  or  even  by  a  machine  (boot  wash 
station), etc. 

The testing requirements for some  specific uses of hard surface disinfectants are discussed 
in separate sections, for example, beehives. 

Disinfection of inner surfaces of pipelines or reservoirs for milk, water or feed for animals are 
considered  food  and  feed  contact  surfaces  and  are  therefore  considered  PT4  (see  section 
5.4.4.3  of  this  Guidance).  Outer  surfaces  of  milking  equipment  are  considered  in  this 
section. 

5.4.3.2.2 Data requirements 

Test methods 

For  efficacy  testing  of  veterinary  hard  surface  biocidal  products,  the  tiered  approach  as 
described in section 5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for a hard surface disinfectant: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a quantitative surface test (phase 2, step 2); 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
porous or non-porous surfaces, contact time, etc.). 

Field  tests  in  phase  3  are  optional,  according  to  section  5.4.0.4.3  of  this  Guidance.  No 
validated test methods are available yet. 

Several methods for testing the efficacy of hard surface disinfectants are available. 

Appendices 2 and 4 give a list of recommended test methods. 

The following documents are recommended for surface disinfection: 

• 

• 

EN 14885: gives an overview of which EN phase 2, step 1 and step 2 tests to use 

for different uses; 

DVG guideline (relevant for testing against endoparasites and virucidal activity on 

porous and non-porous surfaces, as long as no EN tests are available). 

The  use  of  the  specified  tests  is  strongly  recommended  where  they  are  relevant  and 
appropriate.  Where  the  tests  are  not  appropriate  to  the  product,  other  tests  can  be  used, 
although a justification for the relevance of the tests used should also be provided. 

Since  OECD  tests  are  not  specified  for  veterinary  use,  they  are  not  specifically 
recommended. 

In the veterinary area  very often rough, porous surfaces have to be  disinfected (i.e. wood, 
concrete,  rough  plastic  materials).  When  tests  for  porous  surfaces  are  available  it  is 
recommended to use these tests for general surface disinfection in veterinary areas. 

 
 
 
 
 
   
 
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For  boot,  tyre,  and  equipment  disinfection  by  immersion  in  a  bath,  information  should  be 
provided  on  how  long  the  efficacy  of  a  bath  can  be  guaranteed  (time  period,  number  of 
boots etc. passing through). Challenging efficacy tests (capacity tests, see  section 5.4.0.4.1 
of  this  Guidance)  should  be  done,  simulating  the  consecutive  challenge  not  only  by  micro-
organisms  but  also  by  soiling.  A  test  with  relevant  organic  soiling  should  be  provided  in 
order to ensure that biocidal product can be challenged successfully with the test organism 
until  the  end  of  the  claimed  period  of  use.  Alternatively,  for  products  with  one  active 
substance  that  can  easily  be  measured,  efficacy  can  be  demonstrated  using  a  field  test  in 
which  the  amount  of  active  substance  is  measured  several  times  during  the  test  period. 
Efficacy (suspension) tests should be provided with the concentration of the product tested 
(in the suspension test) and the active substance concentration obtained in the field at the 
end of the claimed period of use. 

A  product  can  be  applied  by  airborne  diffusion  of  an  aerosol,  a  smoke,  a  vapour  or  a  gas, 
with the intention to disinfect the surfaces of the walls, floor and ceiling of the room, as well 
as external surfaces  of  the furniture and equipment present in the treated room. For these 
products  the  test  methods  are  described  in  section  5.4.2.5  of  this  Guidance.  These  tests 
should be adapted to fit the conditions (soiling, etc. see section 5.4.3.2.2 of this Guidance) 
for veterinary use. 

When  efficacy  against  a  biofilm  is  claimed,  a  simulated-use  test  or  field  test  has  to  be 
performed, along with a phase 2, step 1 test. See section 5.4.2.11 of this Guidance for test 
methods. 

Where  no  phase  2,  step  2  or  phase  3  tests  are  provided  this  must  be  justified  in  the 
application for authorisation and will be evaluated on a case-by-case basis. 

The EN tests are strongly recommended where available and appropriate. For an overview of 
available EN tests see Appendices 2 and 4. 

Test organisms 

Relevant groups of organisms to be controlled in the veterinary area can be bacteria, yeasts, 
fungal spores, viruses, mycobacteria, bacterial spores, and endoparasites (oocysts). 

Veterinary  hard  surface  biocidal  products  should  be  at  least  sufficiently  effective  against 
bacteria and yeasts. Efficacy tests with these organisms should always be provided. 

Products  for  disinfection  of  veterinary  instruments  and/or  animal  transportation  vehicles 
should not only be effective against bacteria and yeasts but also against viruses. 

Activity against fungi is also required for products used in hatcheries. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. 

The test organisms used in efficacy tests are normally stated in the applicable standard test 
methods. An overview of reference test organisms is given in Appendix 3. 

Test conditions 

It is important that the tests are performed using the same contact time as claimed on the 
label. 

The claimed contact time has to be a realistic value, for instance: 

• 

for  surface  disinfection  products  used  on  the  outside  of  animal  transport  vehicles 
(specifically tyres) the contact time should not exceed 5 minutes; 

 
   
 
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• 

• 

• 

for disinfectants used on boots applied by spraying or walk-through bath the contact 
time should not exceed 1 minute; 

for disinfectants applied by dipping in bath, used on boots, materials etc. the contact 
time should be as claimed on the label; 

for  surface  disinfection  products  used  in  animal  housing  on  floors,  walls  etc.  the 
contact times as stated in the standard tests should be taken into account. 

Additional  contact  times  can  be  considered  if  appropriate  and  justified  by  the  application 
(e.g. overnight disinfection). 

Tests  should  be  carried  out  with  soiling  for  clean  or  dirty  conditions  (low  or  high-level 
soiling)  in  accordance  with  the  test  requirements.  Tests  under  clean  conditions  will  only 
suffice  when  the  label  instructions  state  that  cleaning  prior  to  disinfection  is  necessary.  If 
this  is  not  stated  on  the  label,  the  test  should  be  done  under  dirty  conditions.  The  soiling 
needed  for  clean  and  dirty  conditions  can  be  found  in  the  relevant  EN  tests  and  are 
referenced  in  Appendix  4.  When  the  test  does  not  state  two  levels  of  soiling  (e.g.  porous 
surface test), the soiling referenced in Appendix 4 should be used. 

Normally PT3 products are tested at 10ºC or below since the temperature in animal housings 
can be low. For some uses higher temperatures are acceptable (e.g. hatcheries). Deviations 
from this temperature requirement must be justified in the application and will be evaluated 
on a case-by-case basis. Any limitations on the temperatures at which the product should be 
used, and for which efficacy has been proven should be stated on the label. 

5.4.3.2.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated-use  or  field  tests  have  been  performed  (using  the  required  test  organisms  and 
test conditions), and when the pass criteria for the tests have been met.  

Where pass criteria are available in the standard test these should be met. For PT3 products 
the required log10 reductions are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.3.3 Disinfection of bee hives and beekeeping equipment 

5.4.3.3.1 Introduction 

Disinfection  of  beehives  is  done  to  prevent  spread  of  diseases  from  one  bee  population  to 
the next.  

Only  disinfection  of  empty  bee  hives  and  beekeeping  equipment,  with  products  without  a 
medicinal  claim,  is  a  biocidal  use  for  general  disinfection.  Products  used  in  beehives  with 
bees, honey and brood combes are veterinary medicinal products. These products are within 
the  scope  of  the  Veterinary  Medicinal  Products  Directive  (2001/82/EC  as  amended  by 
2004/28/EC) and are therefore excluded from the BPR. 

Important disease which can be spread via bee hives are American foulbrood (Paenibacillus 
larvae),  European  foulbrood,  (Melissococcus  plutonius),  Nosema  (Nosema  apis,  Nosema 
ceranae),  chalkbrood  (Ascosphaera  apis)  stonebrood  (Aspergillus  flavus)  and  some  viral 
diseases.  Of  these  diseases  American  foulbrood,  which  is  an  endospore-forming  bacterium, 
is the most difficult to control. 

 
 
 
 
 
   
 
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Normal practice in case of American and European foulbrood is to  clean/disinfect bee hives 
and beekeeping equipment and additionally disinfected by scorching with a blowtorch. 

5.4.3.3.2 Data requirements 

Test methods 

For efficacy testing of disinfection products for beekeeping, the tiered approach as described 
in section 5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for disinfectants for bee hives: 

•  a quantitative suspension test (phase 2, step 1; 

•  and a quantitative carrier test (phase 2, step 2) for porous surfaces; 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, etc.). 

Field  tests  in  phase  3  are  optional,  according  to  section  5.4.0.4.3  of  this  Guidance.  No 
validated test methods are available yet. 

There  are  no  standard  tests  available  specifically  for  use  in  bee  hives.  Phase  2,  step  1  EN 
tests for veterinary area are suitable, and for sporicidal activity the EN 13704. EN phase 2, 
step  2  tests  for  veterinary  area  on  porous  material  would  be  suitable  but  they  are  not 
available for all organisms yet. This can be either EN 16437 phase 2, step 2 test on bacteria 
for veterinary area on porous material or DVG guidelines on rough surfaces. These tests can 
be adapted for other organisms. 

In these tests a reference substance must be included. 

Where  no  phase  2,  step  2  tests  for  veterinary  area  on  porous  material  are  available,  the 
available test should be adapted for this use (e.g. EN 16437 adapted for other organisms). 

When  the  claim  for  the  product  is  to  replace  both  the  cleaning/disinfection  step  and  the 
flaming with a welding torch, a field trial has to be provided in which it is demonstrated that 
the  product  is  as  efficacious  against  foulbrood  infected  hives  as  is  cleaning  with  sodium 
hydroxide combined with scorching with a blowtorch. 

For an overview of available EN tests see Appendices 2 and 4. 

Test organisms 

Disinfection  products  for  bee  hives  should  be  at  least  sufficiently  effective  against  bacteria 
and bacterial spores. Efficacy tests with these organisms should always be provided. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. 

For  bacterial  spores  only  a  test  for  the  food  area  is  available  (EN  13704).  For  disinfection 
products for beehives spores  of two bacterial species should be tested. Next to the current 
standard test organism, Bacillus subtilus spores, also Bacillus cereus should be tested. 

Test conditions 

It is important that the tests are performed using the same contact time as claimed on the 
label. 

The claimed contact time has to be a realistic value. 

It  must  be  ensured  that  the  disinfected  parts  stay  wet  during  the  contact  time.  When 
residual efficacy is claimed for dried products this should be demonstrated in efficacy tests. 

 
   
 
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For  disinfection  of  bee  hives  and  beekeeping  equipment,  tests  should  be  performed  under 
dirty conditions (high-level soiling) used for surfaces in the veterinary area. If bee hives are 
not  cleaned  before  disinfection  the  high-level  soiling  for  suspension  tests  should  be  used, 
also in the porous surface test and tests adopted from other areas of use (e.g. EN 13704). 

The soiling needed for dirty conditions can be found in the relevant EN tests and referenced 
in Appendix 4. 

For disinfection of bee hives a temperature of 10ºC or lower is acceptable. Deviations from 
this temperature requirement must be justified in the application and will be evaluated on a 
case-by-case basis. 

5.4.3.3.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated-use  or  field  tests  have  been  performed  (using  the  required  test  organisms  and 
test conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT3 products 
the required log10 reductions are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.3.4 Animal feet disinfection 

5.4.3.4.1 Introduction 

Animal feet disinfection includes hoof and  claw disinfection.  Products  are applied in  a bath, 
through  which  the  animals  can  walk,  or  as  wipes,  foam,  spray,  etc.  See  section  5.4.3.1  of 
this Guidance for overlap with other EU directives. 

5.4.3.4.2 Data requirements 

Test methods 

For efficacy testing of animal feet disinfection products, the tiered approach as described in 
section 5.4.0.4.1 of this Guidance is preferred.  

The following tests are normally required for an animal feet disinfectant: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a quantitative carrier test (phase 2, step 2); 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, etc.). 

Field  tests  in  phase  3  are  optional,  according  to  section  5.4.0.4.3  of  this  Guidance.  No 
validated test methods are available yet. 

There are no standard tests available specifically for use on animal feet. Phase 2, step 1 EN 
tests for veterinary area are suitable. Since hoofs are made of porous material EN phase 2, 
step  2  tests  for  veterinary  area  on  porous  material  would  be  suitable  but  these  are  not 
available for all organisms yet. Alternatively, DVG guideline tests on rough surfaces  can be 
used. 

The phase 2, step 2 test design must always reflect the application. When no standard test 
is used the test design should be discussed with, and agreed by, the CA before testing takes 
place. 

 
 
 
 
 
   
 
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When  no  phase  2,  step  2  or  phase  3  tests  are  provided  this  must  be  justified  in  the 
application and will be evaluated on a case-by-case basis. 

For disinfection in a hoof bath, information should be provided on how long the efficacy of a 
hoof bath can be guaranteed (time period, number of animals passing through). Challenging 
efficacy  tests  (capacity  tests,  see  section  5.4.0.4.1  of  this  Guidance)  should  be  done, 
simulating the consecutive challenge not only by micro-organisms but also by soiling. A test 
with relevant organic soiling should be provided in order to ensure that the biocidal product 
can be challenged successfully with the test organism until the end of the claimed period of 
use.  When  a  challenge  test  is  provided  the  quantitative  suspension  test  can  be  waived. 
Alternatively,  for  products  with  one  active  substance  that  can  easily  be  measured,  efficacy 
can be demonstrated using a field test in which the amount of active substance is measured 
several times during the test period. Efficacy (suspension) tests should be provided with the 
concentration  of  the  product  tested  (in  the  suspension  test)  and  the  active  substance 
concentration obtained in the field at the end of the claimed period of use. 

For an overview of available EN tests see Appendices 2 and 4. 

Test organisms 

Animal  feet  disinfection  should  be  at  least  sufficiently  effective  against  bacteria.  Efficacy 
tests with these organisms should always be provided. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. 

Test conditions 

It is important that the tests are performed using the same contact time as claimed on the 
label. 

The claimed contact time has to be a realistic value, therefore maximum contact times are 
set. 

For animal feet disinfection products the contact time should not exceed 5 minutes. 

It  must  be  ensured  that  it  is  possible  to  keep  the  disinfected  parts  wet  during  the  contact 
time  in  practice.  When  residual  efficacy  is  claimed  for  dried  products  this  should  be 
demonstrated in efficacy tests. 

Tests  should  be  carried  out  with  high-level  soiling  conditions  in  accordance  with  the  test 
requirements.  Soiling  conditions  for  animal  feet  disinfectants  are  the  same  as  for  other 
veterinary area disinfectants. The soiling needed for clean and dirty conditions can be found 
in the relevant EN tests and referenced in Appendix 4. 

Normally animal feet disinfection products are tested at 10ºC since feet disinfection is often 
carried  out  outside animal housings at low temperatures. Deviations from this temperature 
requirement  must  be  justified  in  the  application  and  will  be  evaluated  on  a  case-by-case 
basis. 

5.4.3.4.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated-use  or  field  tests  have  been  performed  (using  the  required  test  organisms  and 
test conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT3 products 
the required log10 reductions are referenced in Appendix 4. 

 
   
 
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Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.3.5 Teat disinfection 

5.4.3.5.1 Introduction 

Teat disinfection products are used to disinfect the teats  of the udder of dairy animals (e.g. 
cows,  sheep  and  goats)  before  or  after  milking.  Products  can  be  applied  by  dipping, 
spraying, foaming, wiping, etc. 

See section 5.4.3.6.1 of this Guidance for overlap with other EU directives. 

5.4.3.5.2 Data requirements 

Test methods 

For efficacy testing of teat disinfection products, the tiered approach as described in section 
5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for a teat disinfectant: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a quantitative carrier test (phase 2, step 2), or a field test; 

all  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, etc.). 

Phase 2, step 1 tests for the veterinary area, with relevant soiling for teat disinfection should 
be used. 

No  European  standard  phase  2,  step  2  tests  are  available  for  teat  disinfection.  To 
demonstrate efficacy a phase 2, step 2 tests should be provided with a test design relevant 
for  the  use.  The  test  design  must  reflect  the  application  and  should  be  discussed  with  and 
agreed by the CA before testing takes place. 

When  standard  tests  become  available,  which  are  relevant  for  teat  disinfectants,  it  is 
recommended to use these tests. 

Alternatively a phase 3 test, field trial, may be provided with a test design relevant for the 
use. The test design must reflect the application, should include a control with water instead 
of biocide, and should be discussed with and agreed by the CA before testing takes place. 

Disinfectant towelettes/wipes 
For  disinfectant  wipes,  the  phase  2,  step  1  tests  should  be  done  preferably  with  the  liquid 
extracted from the wipe or if difficult to extract, use the liquid as it is before it is added to 
the wipes. Phase 2, step 2 tests should be tests with mechanical action or, when this test is 
not available, with liquid extracted from the wipe (not the original liquid), with a justification 
of the volume that is applied per square centimetre. In addition, a test must be performed 
that  shows  that  either  the  wipe  will  still  disinfect  after  the  wipe  dries  out  or  that  the  wipe 
stays wet long enough to disinfect according to the claim. In addition, the use directions can 
address these issues, for instance, stating on the label that only wet wipes are efficacious, or 
giving expiry dates for re-sealable packages. 

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Example of phase 2, step 2 tests 
The  phase  2,  step  2  surface  carrier  test  can  be  derived  from  adaptation  of  CEN  TC  216 
surface tests. Instead of a hard surface carrier, carriers involved could be made of material 
simulating the teat. Justification for the used carrier should be provided. 

Cells  of  test  organisms  should  be  applied  and  fixed  onto  the  surface  in  a  manner  which 
represents  pre-  and  post-application,  (dried  in  case  of  pre-milking  or  not  dried  in  case  of 
post-milking), and incubated with the product for the appropriate time (see EN phase 2, step 
2  test,  for  example,  EN  14349  or  EN  16437,  for  growth  conditions,  controls,  etc.).  After 
incubation  with  the  product  the  cell  count  reduction  is  evaluated  and  compared  to  a  water 
control. 

The test design should be discussed with and agreed by the CA before testing takes place. 

Test organisms 

Teat  disinfection  products  should  be  at  least  sufficiently  effective  against  bacteria  and 
yeasts. Efficacy tests with these organisms should always be provided. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is intended to be claimed. 

Virucidal activity 

For products used as teat disinfectants a differentiation in the virucidal activity is made. 

The claims can be: 

• 

full virucidal activity or 

•  activity against enveloped viruses. 

For each claim different test organisms should be tested. 

The EN 14675 test for virucidal activity in the veterinary area tests Bovine Enterovirus Type 
1  (ECBO),  a  non-enveloped  virus.  When  this  test  is  passed,  full  virucidal  activity  can  be 
claimed. 

Activity  against  enveloped  viruses  can  be  claimed  when  MVA  =  Modified  Vacciniavirus 
Ankara is tested in a (modified) EN 14675 test. 

When  only  activity  against  enveloped  viruses  is  demonstrated  the  label  claim  cannot  be 
“virucidal”.  The  SPC  should  clearly  state  which  of  the  possible  virucidal  claims  was 
demonstrated. 

Test conditions 

It is important that the tests are performed using the same contact time as claimed on the 
label. 

The claimed contact time has to be a realistic value, therefore maximum contact times are 
set. 

For post-milking teat disinfection products the contact time is normally 1 minute but should 
not exceed 5 minutes. 

The  contact  time  for  pre-milking  teat  disinfection  products  is  normally  30  seconds  or  less 
and should not exceed 60 seconds. 

Deviations  from  this  contact  time  requirement  must  be  justified  in  the  application  for 
authorisation and will be evaluated on a case-by-case basis. 

 
   
 
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Tests for pre-milking products should be carried out with either low or high-level soiling for 
veterinary surfaces, depending on the instructions given for pre-cleaning procedures. 

Tests  for  post-milking  products  should  be  carried  out  with  soiling  for  teat  disinfectants  in 
accordance  with  the  test  requirements.  Soiling  conditions  for  teat  disinfectants  are 
mentioned in the bactericidal test and  should  be used for the test with other organisms as 
well. 

The soiling needed can be found in EN 1656 and referenced in Appendix 4. 

For teat disinfection a test temperature of 30ºC or lower is acceptable. Deviations from this 
temperature  requirement  must  be  justified  in  the  application  and  will  be  evaluated  on  a 
case-by-case basis. 

5.4.3.5.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated-use  or  field  tests  have  been  performed  (using  the  required  test  organisms  and 
test conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT3 products 
the required log10 reductions are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.3.6 Other animal corporal hygiene 

5.4.3.6.1 Introduction 

Disinfectants  for  animal  corporal  hygiene  are  used  to  disinfect  the  skin  of  animals.  This 
section includes all animal skin disinfectants,  which are not  covered in the sections on teat 
or animal feet disinfection below. 

A  product  applied  on  animal  skin  could  be  either  a  biocidal  or  a  veterinary  medicinal  or  a 
product  for  cleaning  or  cosmetic  purposes.  If  the  product  under  investigation  is  within  the 
scope  of  the  Veterinary  Medicinal  Products  Directive  (2001/82/EC  as  amended  by 
2004/28/EC)  it  is  excluded  from  the  BPR  for  the  respective  use.  When  a  product  does  not 
have  a  biocidal  claim  (e.g.  skin  disinfection,  activity  against  micro-organisms  claimed)  but 
only  a  cosmetic  claim  (e.g.  cleaning  skin,  paws)  it  is  excluded  from  the  BPR  for  the 
respective use. 

Products  for  disinfection  of  damaged  skin  (e.g.  wound  disinfection)  or  disinfection  of 
undamaged  skin  before  a  medical  treatment  (e.g.  pre-operative  skin  disinfection  or 
disinfection before injection) are always veterinary medicinal products. 

When applying for authorisation for an animal corporal hygiene biocidal product within PT3 a 
detailed  description  of  the  intended  use  should  be  given,  to  prevent  authorisation  of 
veterinary  medicinal  products  or  medicinal  uses,  as  biocides  (e.g.  the  claim  “animal  skin 
disinfection” is insufficient). 

For  products  that  fall  under  the  BPR  the  data  requirements  described  in  the  following 
sections apply. 

 
 
 
 
 
   
 
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5.4.3.6.2 Data requirements 

Test methods 

For efficacy testing of animal corporal hygiene products, the tiered approach as described in 
section 5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for an animal corporal hygiene disinfectant: 

•  a quantitative suspension test (phase 2, step 1; 

•  and a quantitative carrier test (phase 2, step 2); 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, etc.). 

Field  tests  in  phase  3  are  optional,  according  to  section  5.4.0.4.3  of  this  Guidance.  No 
validated test methods are available yet. 

Phase 2, step 1 tests for the veterinary area can be used. 

No  European  standard  phase  2,  step  2  tests  are  available  for  animal  skin  disinfection.  To 
demonstrate efficacy a phase 2, step 2 tests should be provided with a test design relevant 
for  the  use.  The  test  design  must  reflect  the  application  and  should  be  discussed  with  and 
agreed by the CA before testing takes place. 

When  standard  tests  become  available,  which  are  relevant  for  skin  disinfectants,  it  is 
recommended to use these tests. 

For an overview of available EN tests see Appendices 4 and 6. 

Example of phase 2, step 2 tests 
The  phase  2,  step  2  surface  carrier  test  can  be  derived  from  adaptation  of  CEN  TC  216 
surface  tests.  Instead  of  a  hard  surface  carrier,  carriers  could  be  made  of  material 
simulating  animal  skin16.  Method  are  currently  being  developed,  but  their  aptitude  for  the 
respective biocidal use/demonstration of efficacy for animal skin disinfectants remains to be 
proven. Justification for the used carrier should be provided. 

Cells  of  test  organisms  could  be  applied  to  the  surface,  dried,  and  incubated  with  the 
product  for  the  appropriate  time  (see  EN  phase  2,  step  2  test,  e.g.  EN 14349,  for  growth 
conditions,  controls,  etc.).  After  incubation  with  the  product  the  cell  count  reduction  is 
evaluated and compared to a water control. 

For an overview of available EN tests see Appendices 2 and 4. 

Test organisms 

Animal  corporal  hygiene  products  should  be  at  least  sufficiently  effective  against  bacteria 
and yeasts. Efficacy tests with these organisms should always be provided. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. 

Test conditions 

It is important that the tests are performed using the same contact time as claimed on the 
label. 

16 Please take into account EU regulation 1069/2009, on animal by-products. 

 
   
 
 
 
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The claimed contact time has to be a realistic value. 

It  must  be  ensured  that  the  test  surface  does  not  remain  wet  longer  than  the  part  of  the 
animal  body  treated  with  the  product,  for  example,  by  using  higher  (more  realistic) 
temperatures.  When  residual  efficacy  is  claimed  this  should  be  demonstrated  in  efficacy 
tests. 

Tests should be carried out with high level or low level soiling conditions in accordance with 
the test requirements. Soiling conditions for animal corporal hygiene products are the same 
as for other  veterinary  area disinfectants.  The  soiling needed  for  clean and dirty  conditions 
can be found in the relevant EN tests and referenced in Appendix 4. 

For  animal  corporal  hygiene  products  a  test  temperature  of  30ºC  or  lower  is  acceptable. 
Deviations from this temperature requirement must be justified in the application and will be 
evaluated on a case-by-case basis. 

5.4.3.6.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated-use  tests  have  been  performed  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT3 products 
the required log10 reductions are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.3.7 Disinfection of hatching-eggs 

5.4.3.7.1 Introduction 

Disinfection  of  hatching-eggs  includes  the  disinfection  of  eggs  before  they  hatch  in 
hatcheries. Products are applied in a bath, as a spray, as wipes, fumigation, etc. 

5.4.3.7.2 Data requirements 

Test methods 

For  efficacy  testing  of  disinfection  products  for  hatching-eggs,  the  tiered  approach  as 
described in section 5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for disinfectant for hatching-eggs: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a quantitative carrier test (phase 2, step 2); 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, etc.). 

Field  tests  in  phase  3  are  optional,  according  to  section  5.4.0.4.3  of  this  Guidance.  No 
validated test methods are available yet. 

Phase 2, step 1 tests for the veterinary area can be used. 

As  long  as  no  standard  phase  2,  step  2  tests  are  available  it  is  not  obligatory  to  provide 
these  tests.  Phase  2,  step  2  tests  have  to  be  provided  as  soon  as  standard  tests  are 
available. 

 
 
 
 
 
   
 
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When  no  phase  2,  step  2  or  phase  3  tests  are  provided  this  must  be  justified  in  the 
application and will be evaluated on a case-by-case basis. 

For egg disinfection in a bath, information should be provided on how long the efficacy of a 
bath can be guaranteed (time period, number of eggs passing through). Challenging efficacy 
tests (capacity tests, see section 5.4.0.4.1 of this Guidance) should be done, simulating the 
consecutive challenge not only by  micro-organisms but also by soiling. A test with relevant 
organic soiling should be provided in order to ensure that biocidal product can be challenged 
successfully  with  the  test  organism  until  the  end  of  the  claimed  period  of  use.  When  a 
challenge test is provided the quantitative suspension test can be waived. Alternatively, for 
products  with  one  active  substance  that  can  easily  be  measured,  efficacy  can  be 
demonstrated using a field test in which the amount of active substance is measured several 
times  during  the  test  period.  Efficacy  (suspension)  tests  should  be  provided  with  the 
concentration  of  the  product  tested  (in  the  suspension  test)  and  the  active  substance 
concentration obtained in the field at the end of the claimed period of use. 

For  products  applied  by  airborne  diffusion  of  an  aerosol,  a  smoke,  a  vapour  or  a  gas,  with 
the intention to disinfect the room, as well as on external surfaces of the eggs in the room, 
the  test  methods  are  described  in  section  5.4.2.5  of  this  Guidance.  These  tests  should  be 
adapted  to  fit  the  conditions  (soiling,  etc.  see  section  5.4.3.7.2  of  this  Guidance)  for 
veterinary use. 

For an overview of available EN tests see Appendices 2 and 4. 

Test organisms 

Disinfection  products  for  hatching-eggs  should  be  at  least  sufficiently  effective  against 
bacteria and fungi. Efficacy tests with these organisms should always be provided. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. 

Test conditions 

It is important that the tests are performed using the  same contact time as claimed on the 
label. 

The claimed contact time has to be a realistic value. 

It  must  be  ensured  that  the  disinfected  parts  stay  wet  during  the  contact  time.  When 
residual efficacy is claimed for dried products this should be demonstrated in efficacy tests. 

Tests should be  carried out with soiling for  clean or dirty conditions in accordance with the 
test requirements. Tests under clean conditions will only suffice when the label instructions 
state  that  cleaning  prior  to  disinfection  is  necessary.  If  this  is  not  stated  on  the  label,  the 
test  should  be  done  under  dirty  conditions.  Soiling  conditions  for  of  hatching-eggs 
disinfectants are the same as for other veterinary area disinfectants. The soiling needed for 
clean and dirty conditions can be found in the relevant EN tests and referenced in Appendix 
4. 

For  disinfection  of  hatching-eggs  a  temperature  of  30ºC  or  lower  is  acceptable.  Deviations 
from this temperature requirement must be justified in the application and will be evaluated 
on a case-by-case basis. 

 
   
 
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5.4.3.7.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  or,  when 
applicable,  simulated-use  or  field  tests  have  been  performed  (using  the  required  test 
organisms and test conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT3 products 
the required log10 reductions are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.3.8 Textile disinfection in PT3 

5.4.3.8.1 Introduction 

Textile disinfection products within PT3 are mainly used to disinfect the cloths used for teat 
cleaning/disinfection of dairy cattle before milking. Products are normally applied by dipping 
the cloth in a disinfectant solution. For other uses the requirements below should be adapted 
to fit the intended use. 

5.4.3.8.2 Data requirements 

Test methods 

For  efficacy  testing  of  textile  disinfection  products,  the  tiered  approach  as  described  in 
section 5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for textile disinfection products: 

•  a quantitative suspension test (phase 2, step 1); 

•  a  quantitative  carrier  test  involving  carriers  made  of  test  fabric  (cotton,  polyester) 

(phase 2, step 2); 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, repeated challenges, etc.). 

Field  tests  in  phase  3  are  optional,  according  to  section  5.4.0.4.3  of  this  Guidance.  No 
validated test methods are available yet. 

Test methods for textile disinfection are described in section 5.4.2.10 of this Guidance.  

Currently, the following tests are available: 

•  phase 2, step 1 suspension tests as described in EN 14885,  

•  phase 2, step 2 tests involving test fabrics in: 

o  a small scale laboratory setting (e.g. ASTM E2406) or; 
o  a full-scale laundry machine test (EN 16616, or DGHM). 

In the phase 2, step 2 tests fabric is contaminated with test organisms and then exposed to 
the  disinfectant.  These  tests  should  be  adapted  to  fit  the  conditions  (soiling,  etc.  see 
4.8.2.3)  for  veterinary  use.  For  disinfection  in  washing  machines  a  full-scale  laundry 
machine test, according to test conditions mentioned in section 5.4.2.10.2 of this Guidance, 
is obligatory. 

The EN tests are strongly recommended where available and appropriate. For an overview of 
available EN tests see Appendices 2 and 4. 

 
 
 
 
 
   
 
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Test organisms 

Textile  disinfection  products  should  be  at  least  sufficiently  effective  against  bacteria  and 
yeasts. Efficacy tests with these organisms should always be provided. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. 

When  the  product  is  intended  to  be  used  at  high  temperatures  (>40  ºC)  relevant  test 
organisms  for  these  temperatures  should  be  used  as  described  in  section  5.4.0.4.4  of  this 
Guidance. 

Test conditions 

It is important that the tests are performed using the same contact time as claimed on the 
label. 

The claimed contact time has to be a realistic value. 

The contact time products intended for disinfection of textile in between milking sessions can 
be several hours. 

Tests should be  carried out with soiling for  clean or dirty conditions in accordance with the 
test requirements for veterinary area. Tests under clean conditions will only suffice when the 
label instructions state that cleaning prior to disinfection is necessary. If this is not stated on 
the  label,  the  test  should  be  done  under  dirty  conditions.  Soiling  conditions  for  milking-
textile disinfectants are the same as for teat disinfectants. The soiling needed for clean and 
dirty conditions can be found in the relevant EN tests and referenced in Appendix 4. 

For textile disinfection a test temperature should be according to the use instructions. When 
the textile is immersed in a bucket with warm water it should be taken into account that the 
water temperature will decrease during the disinfection process. This should be reflected in 
the test conditions. 

5.4.3.8.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated-use  or  field  tests  have  been  performed  (using  the  required  test  organisms  and 
test conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT3 products 
the required log10 reductions are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.3.9 Disinfection of manure, litter and other substrates for veterinary use 

5.4.3.9.1 Introduction 

Manure  mainly  consists  of  urines  and  faeces  (organic  matters  and  intestinal  bacteria)  in 
which  can  also  be  mixed  straw  of  litters  in  more  or  less  big  quantity,  according  to  the 
breeding technique (partial slats or complete slats). 

Manure  has  a  potential  for  spreading  infectious  diseases  and  biocidal  products  are  used  to 
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Litters are usually used in animal housing (poultry, pigsties, etc.) and also for pets in private 
uses.  They  absorb  urines  and  faeces.  Biocidal  products  are  mainly  used  to  deodorize  and 
neutralize bad smells. 

5.4.3.9.2 Data requirements 

Test methods 

For efficacy testing of disinfects biocidal products used for manure and litter disinfection, the 
tiered approach as described in section 5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required: 

•  a quantitative suspension test (phase 2, step 1), 

•  and simulated-use test, or field test 

all  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, mode of application, pH, etc.). 

An example of a simulated-use test could be autoclaved manure or litter collected in animal 
housing and tested in the lab with inoculation of target organisms. A control without addition 
of disinfectants should be included. The test design should be discussed with and agreed by 
the CA before testing takes place. 

In case of products claiming malodour control, the same requirements as mentioned in the 
section 5.4.0.5.4 of this Guidance, are required. 

Test organisms 

Generally, target organisms have to be representative of the veterinary area, as stated in EN 
14885. 

For specific uses in industry, an exception can be made when sound justification is provided. 
This will be evaluated on a case-by-case basis. 

Taking into account the specificity of some kind of uses, it may be justified to test additional 
target  organisms  (e.g.  Brachyspira  hyodysenteriae  agent  of  swine  dysentery,),  special 
growth conditions, etc. 

In  case  of  malodour  control,  tests  should  be  performed  with  odour  producing  micro-
organisms.  A  justification  for  which  bacteria,  fungi,  etc.  are  relevant  to  the  intended  use 
should be provided. Along with these laboratory tests, an odour test can be performed. 

Test conditions 

It  is  important  that  the  tests  are  performed  with  the  same  contact  time  as  claimed  on  the 
label. 

The claimed contact time has to be a realistic value. 

Quantitative  suspension  tests  must  be  carried  out  with  high  level  soiling  conditions  and  a 
temperature of 10°C or less. 

The  test  temperature  should  be  according  to  the  use  instructions  on  the  label  and 
appropriate to the uses (stables, private homes, etc.). 

Field  and  simulated-use  test  have  to  be  performed  according  to  the  dose,  conditions  and 
mode  of  application  of  the  product.  For  example,  if  the  product  is  applied  on  top  of  the 
manure, the product does not have to be mixed with the organic matter but has to be put on 
top  of  it  (to  mimic  the  diffusion  and  evaluate  efficacy  in  the  same  conditions  as  in  the 
practice). 

 
 
 
 
 
   
 
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In case  of litter, if persistence is claimed with  some  recommendations about the  frequency 
of  renewal,  adequate  simulating  tests  (with  appropriate  contribution  of  organic  matters  in 
the test) have to be performed. 

Deviations from these requirements must be justified in the application for authorisation and 
will be evaluated on a case-by-case basis. 

5.4.3.9.3 Acceptance criteria 

A product will be assessed to be sufficiently effective if the required laboratory and, field (or 
simulated-use)  tests  have  been  performed  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT3 products 
the required log10 reductions are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.3.10 Other uses in PT3 

Several  uses  of  PT3  products  have  been  specified  in  the  above  sections  and  data 
requirements and acceptance  criteria for these uses are described. For products with  other 
uses  that  do  not  fit  in  one  of  the  described  uses,  it  is  up  to  the  applicant  to  demonstrate 
efficacy in an appropriate way. 

In  general,  the  tiered  approach  as  described  in  section  5.4.0.4.1  of  this  Guidance  is 
preferred. Where possible the standard tests required for the described uses should be taken 
(e.g.  EN  phase  2,  step  1  and  step  2  tests  for  veterinary  area).  Where  the  tests  are  not 
appropriate  for  the  product,  other  tests  can  be  used.  In  that  case,  a  justification  for  the 
relevance  of  the  tests  used  should  be  provided.  The  test  design  should  be  discussed  with 
and agreed by the CA before testing takes place. The evaluation will be done on a case-by-
case basis by the CAs. 

The guidance will be updated when new methods become available. 

5.4.4 PT4 Food and feed area disinfectants 

5.4.4.1 Introduction 

Product type 4 contains biocidal products used for the disinfection of equipment, containers, 
consumption  utensils,  surfaces  or  pipework  associated  with  the  production,  transport, 
storage or consumption of food or feed (including drinking water) for humans and animals. 

Some  disinfectants  applied  in  the  food  or  feed  area  can  be  either  biocidal  product  or  a 
preservative  for  food  or  feed.  If  the  product  under  investigation  is  within  the  scope  of 
Regulations  (EC)  852/2004,  853/2004  and  854/2004  on  food  hygiene,  it  is  excluded  from 
the BPR. The Regulation 852/2004 is on the hygiene of foodstuffs; the Regulation 853/2004 
lays  down  specific  hygiene  rules  for  food  of  animal  origin;  the  Regulation  854/2004  lays 
down  specific  rules  for  the  organisation  of  official  controls  on  products  of  animal  origin 
intended for human consumption. 

In  the  sections  below  the  requirements  and  acceptance  criteria  for  most  common  uses  are 
specified. For other uses and claims that are not specifically mentioned the requirements will 
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5.4.4.2 Disinfection of hard surfaces in food and feed area PT4 

5.4.4.2.1 Introduction 

Biocides  can  be  used  to  disinfect  hard  surfaces  in  areas  such  as  food  industry,  kitchens  in 
restaurants  or  homes,  shops  like  butchers  and  grocery  shops  were  food  is  processed  etc. 
These  surfaces  may  be  tables,  floors,  walls,  the  outsides  of  machinery,  equipment, 
reservoirs  for  water  or  feed  in  animal  housing  etc.  Products  are  often  wiped,  sprayed, 
foamed, applied by low to high pressure etc., onto the surface, and maybe washed or wiped 
off after a certain contact time. 

The testing requirements for some  specific uses of hard surface disinfectants are discussed 
in separate sections, for example, CIP, equipment and dishwashing disinfectants etc. 

5.4.4.2.2 Data requirements 

Test methods 

For efficacy testing of food and feed area biocidal products used on hard surfaces, the tiered 
approach as described in section 5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for a hard surface disinfectants: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a quantitative surface test (phase 2, step 2); 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, etc.). 

Tests in phase 3 are optional, according to  section 5.4.0.4.3 of this Guidance. No validated 
test methods are available yet. 

Several  methods  for  testing  the  efficacy  of  hard  surface  disinfectants  are  available.  Tests 
with  mechanical  action  might  be  adopted  from  medical  area,  if  appropriate.  Appendices  2 
and 4 give a list of recommended test methods. 

The following documents are recommended for surface disinfection: 

•  EN  14885:  gives  an  overview  of  which  EN  phase2/step1  and  step2  tests  to  use  for 

different uses; 

if CEN standards are not relevant or available for the use or organisms claimed the following 
documents are recommended if appropriately reflecting the application: 

•  OECD  guidance  for  the  testing  of  chemicals:  Quantitative  method  for  evaluating 
activity  of  microbiocides  used  on  hard  non-porous  surfaces  (these  are  surface  tests 
which would be considered phase 2, step 2 tests). 

The  use  of  the  specified  tests  is  strongly  recommended  where  they  are  relevant  and 
appropriate. 

When efficacy against biofilm is claimed a simulated-use test or field test has to be provided, 
next to a phase 2, step 1 test. See section 5.4.2.11 of this Guidance for test methods. 

A  product  can  be  applied  by  airborne  diffusion  of  an  aerosol,  a  smoke,  a  vapour  or  a  gas, 
with the intention to disinfect on the surfaces of the walls, floor and ceiling of the room, as 
well as on external surfaces of the furniture and equipment present in the treated room. For 
these  products  the  test  methods  are  described  in  section  5.4.2.5  of  this  Guidance.  These 
tests  should  be  adapted  to  fit  the  conditions  (soiling,  etc.  see  section  5.4.3.2.2  of  this 
Guidance) for use in food and feed area. 

 
 
 
 
 
   
 
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Disinfectant towelettes/wipes  

For  disinfectant  wipe,  the  phase  2,  step  1  tests  should  be  done  preferably  with  the  liquid 
extracted from the wipe, or if difficult to extract, use the liquid as it is before it is added to 
the  wipes.  Phase  2,  step  2  tests  should  be  tests  with  mechanical  action.  These  tests  are 
available for bacteria and yeasts. For testing other organisms surface tests can be done with 
liquid extracted from the wipe (not the original liquid), with a justification of the volume that 
is  applied  per  square  centimetre.  In  addition,  a  test  must  be  performed  that  shows  that 
either  the  wipe  will  still  disinfect  after  the  wipe  dries  out  or  that  the  wipe  stays  wet  long 
enough to disinfect according to the claim. In addition, the use directions can address these 
issues,  for  instance,  stating  on  the  label  that  only  wet  wipes  are  efficacious,  defining  the 
surface  area  each  towel  can  disinfect  (e.g.  0.5  m2),  or  giving  expiry  dates  for  re-sealable 
packages. 

Test organisms 

Food and feed hard surface biocidal products should be at least sufficiently effective against 
bacteria and yeasts. Efficacy tests with these organisms should always be provided. 

For specific uses in industry, an exception can be made when sound justification is provided. 
This will be evaluated on a case-by-case basis. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. Relevant groups of test organisms, next to bacteria and yeasts, can be 
fungi  (fungal  spores),  viruses,  bacteriophages,  and  bacterial  spores.  Bacteriophages  are 
mainly of importance in the dairy industry. 

The test organisms used in efficacy tests are normally stated in the applicable standard test 
methods.  For  food  area  disinfectants  Salmonella  Thyphimurium,  Salmonella  spp.,  Listeria 
spp.  and  Campylobacter  jejuni  are  relevant  target  organisms.  For  products  which  claim 
general  efficacy  against  bacteria,  the  standard  test  bacteria  should  be  tested.  For  these 
products  efficacy  against  Salmonella  spp.,  Listeria  spp.  and  Campylobacter  jejuni  is 
assumed, because they are more susceptible than the standard test bacteria. 

The  EN  standards  for  food  area  only  include  a  test  on  bacteriophages  but  not  on  other 
viruses. To demonstrate a general virus  claim a modified EN phase 2,  step 1 test (medical 
area  test  with  food  area  soiling)  can  be  provided  with  Adenovirus  and  Murine  Norovirus  as 
test organism and a phase 2, step 2 test (either modified EN medical test, or DVG test or, as 
soon as available, an EN food area test) with Murine Norovirus. 

An overview of reference test organisms is given in Appendix 3. 

Test conditions 

It  is  important  that  the  tests  are  performed  with  the  same  contact  time  as  claimed  on  the 
label. The claimed contact time has to be a realistic value. 

Tests should be  carried out with soiling for  clean or dirty conditions in accordance with the 
test requirements. Tests under clean conditions will only suffice when the label instructions 
state that cleaning prior to disinfection is necessary. If this is not stated on the label the test 
should be done under dirty conditions. Note that for use in specific industries different types 
of soiling for dirty conditions should be used. 

The  soiling  needed  for  clean  and  dirty  conditions  can  be  found  in  the  relevant  EN  tests  or 
EN 14885 (version 2014 or later) and referenced in Appendix 4. 

 
   
 
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If  a  product  is  intended  to  be  used  in  more  than  one  area  of  use  (e.g.  milk  industry  and 
meat  industry)  it  is  justified,  after  having  identified  the  most  challenging  test  organism,  to 
test the relevant soiling types with this organism. That applies only per group of organisms 
(e.g. bacteria). 

The test temperature should be according to the use instructions on the label. Food and feed 
area disinfectants are generally used at room temperature (test temperature 20 ºC) but for 
some uses and claims (e.g. surfaces in cold storage rooms) low temperatures of 4 ºC or 10 
ºC are relevant and should be tested. 

5.4.4.2.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated-use  or  field  tests  have  been  performed  (using  the  required  test  organisms  and 
test conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT4 products 
the required log10 reductions are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.4.3 Disinfection of inner surfaces in PT4 

5.4.4.3.1 Introduction 

Biocides can be used to disinfect the inner surfaces of pipes, tanks, fillers, mixers, and other 
machines which come in contact with food or feed (including liquids). This includes food and 
feed  industry,  milking  equipment  on  farms,  large  equipment  in  restaurants  or  shops  were 
food  is  processed,  etc.  Inner  surfaces  in  contact  with  water  are  discussed  in  the  following 
sections. 

These  surfaces  are  disinfected  by  filling  and  circulating  the  biocide  in  the  pipes,  tanks, 
machines, etc. with disinfectant (Cleaning In Place, CIP). Also disinfection of inner surfaces 
of equipment by filling without circulation (not using CIP) is included in this section. 

5.4.4.3.2 Data requirements 

Test methods 

For efficacy testing of food and feed area biocidal products used on inner surfaces using CIP, 
the following tests are normally required:  

•  quantitative  suspension  tests  (phase  2,  step  1),  simulating  practical  conditions 
appropriate to its intended use (temperature, soiling, different surfaces, contact time, 
etc.). 

For efficacy testing of food and feed area biocidal products used on inner surfaces by filling 
without circulation, the following tests are normally required for these disinfectants: 

•  quantitative suspension tests (phase 2, step 1); 

•  and a quantitative surface test (phase 2, step 2); 

both  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
different surfaces, contact time, etc.). 

Tests in phase 3 are optional, according to  section 5.4.0.4.3 of this Guidance. No validated 
test methods are available yet. 

 
 
 
 
 
   
 
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Several  methods  for  testing  the  efficacy  of  inner  surface  disinfectants  are  available. 
Appendices 2 and 4 give a list of recommended test methods. 

The following documents are recommended for inner surface disinfection using CIP: 

•  EN 14885 gives an overview of which EN phase 2, step 1 and step 2 tests to use for 

different uses; 

if CEN standards are not relevant or available for the use or organisms claimed the following 
documents are recommended if appropriately reflecting the application: 

•  OECD  guidance  for  the  testing  of  chemicals:  Quantitative  method  for  evaluating 
activity of microbiocides used on hard non-porous surfaces. (These are surface tests 
which would be considered phase 2, step 2 tests). 

The  use  of  the  specified  tests  is  strongly  recommended  where  they  are  relevant  and 
appropriate. 

When efficacy against biofilm is claimed a simulated-use test or field test has to be provided, 
next to a phase 2, step 1 test. See section 5.4.2.11 of this Guidance for test methods. 

When the disinfection is done with vaporised biocide a simulated-use test or a field test has 
to be provided. See section 5.4.2.5 of this Guidance for test methods. 

Test organisms 

Food and feed hard surface biocidal products should be at least sufficiently effective against 
bacteria and yeasts. Efficacy tests with these organisms should always be provided. 

For specific uses in industry, an exception can be made when sound justification is provided. 
This will be evaluated on a case-by-case basis. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms  is  claimed.  Relevant  groups  of  organisms,  next  to  bacteria  and  yeasts,  can  be 
fungal spores, viruses, phages, and bacterial spores. Phages are mainly of importance in the 
dairy industry. 

The test organisms used in efficacy tests are normally stated in the applicable standard test 
methods. For food area disinfectants Salmonella spp., Listeria spp. and Campylobacter jejuni 
are  relevant  target  organisms.  For  products  which  claim  general  efficacy  against  bacteria, 
the standard test bacteria should be tested. For these products  efficacy against  Salmonella 
spp., Listeria spp. and Campylobacter jejuni is assumed, because they are more susceptible 
than the standard test bacteria. 

The  EN  standards  for  food  area  only  include  a  test  on  bacteriophages  but  not  for  other 
viruses. To demonstrate a general virus  claim a modified EN phase 2,  step 1 test (medical 
area  test  with  food  area  soiling)  can  be  provided  with  Adenovirus  and  Murine  Norovirus  as 
test organism and a DVG phase 2, step 2 test. 

When  CIP  is  done  at  high  temperatures  relevant  test  organisms  for  these  temperatures 
should be used as described in section 5.4.0.4.4 of this Guidance. 

An overview of test organisms, also for high temperatures, is given in Appendix 3. 

Test conditions 

It  is  important  that  the  tests  are  performed  with  the  same  contact  time  as  claimed  on  the 
label. The claimed contact time has to be a realistic value. 

Tests should be  carried out with soiling for  clean or dirty conditions in accordance with the 
test requirements. Tests under clean conditions will only suffice when the label instructions 

 
   
 
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state that cleaning prior to disinfection is necessary. If this is not stated on the label the test 
should be done under dirty conditions. Note that for use in specific industries different types 
of soiling for dirty conditions should be used. 

The soiling needed for clean and dirty conditions can be found in the relevant EN tests or EN 
14885 (version 2014 or later) and referenced in Appendix 4. 

The test temperature should be according to the use instructions on the label. Food and feed 
area disinfectants are generally used at room temperature (test temperature 20 ºC) but for 
some  uses  and  claims  other  temperatures  are  relevant.  For  example,  for  surfaces  in  cold 
machinery,  low  temperatures  of  4  ºC  or  10  ºC  are  relevant  and  should  be  tested.  CIP 
disinfection is often done at high temperatures of 40 to 80 ºC. When this is the intended use 
the  test  temperature  should  be  in  accordance  with  the  use  and  relevant  test  organisms 
should be used (see section 5.4.4.3.2 of this Guidance). 

5.4.4.3.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and,  when 
applicable,  simulated-use  or  field  tests  have  been  performed  (using  the  required  test 
organisms and test conditions), and when the pass criteria for the tests have been met. 

Where  pass  criteria  are  available  in  the  standard  tests  these  should  be  met.  For  PT4 
products the required log10 reductions tests are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.4.4 Equipment disinfection by soaking 

5.4.4.4.1 Introduction 

Biocides can be used to disinfect dishes, equipment, crates, boxes, etc. by soaking. This can 
include  dishwashing  disinfectants,  however,  normal  dishwashing  detergents  are  cleaning 
products  and  not  included  in  the  BPR.  Equipment  disinfection  in  washing  machines  is 
covered in the next section. 

This can be used in areas such as food industry, kitchens in restaurants or homes, shops like 
butchers and grocery shops were food or feed is processed, etc. 

5.4.4.4.2 Data requirements 

Test methods 

For  efficacy  testing  of  equipment  and  dish  washing  disinfectants  the  tiered  approach  as 
described in section 5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for these disinfectants: 

•  a quantitative suspension test (phase 2, step 1; 

•  and a quantitative surface test (phase 2, step 2); 

both  tests  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature, 
soiling, different surfaces, contact time, etc.). 

Tests in phase 3 are optional, according to  section 5.4.0.4.3 of this Guidance. No validated 
test methods are available yet. 

Several methods for testing the efficacy of hard surface disinfectants are available. 

Appendices 2 and 4 give a list of recommended test methods. 

 
 
 
 
 
   
 
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The following documents are recommended for equipment and dish washing disinfection: 

•  EN  14885:  gives  an  overview  of  which  EN  phase2/step1  and  step2  tests  to  use  for 

different uses, 

if  CEN  standards  are  not  relevant  or  not  available  for  the  use  or  organisms  claimed  the 
following documents are recommended if appropriately reflecting the application: 

•  OECD  guidance  for  the  testing  of  chemicals:  Quantitative  method  for  evaluating 
activity of microbiocides used on hard non-porous surfaces. (These are surface tests 
which would be considered phase 2, step 2 tests) 

The  use  of  the  specified  tests  is  strongly  recommended  where  they  are  relevant  and 
appropriate. 

When efficacy against biofilm is claimed a simulated-use test or field test has to be provided, 
next to a phase 2, step 1 test. See section 5.4.2.11 of this Guidance for test methods. 

Test organisms 

Equipment  and  dish  washing  disinfectants  should  be  at  least  sufficiently  effective  against 
bacteria and yeasts. Efficacy tests with these organisms should always be provided. 

For specific purposes in industrial uses, an exception can be made when sound justification 
is provided. This will be evaluated on a case-by-case basis. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. Relevant groups of test organisms, next to bacteria and yeasts, can be 
fungi  (fungal  spores),  viruses,  bacteriophages,  and  bacterial  spores.  Bacteriophages  are 
mainly of importance in the dairy industry. 

The test organisms used in efficacy tests are normally stated in the applicable standard test 
methods.  For  dish  washing  disinfectants  Salmonella  spp.,  Listeria  spp.  and  Campylobacter 
jejuni  are  relevant  target  organisms.  For  products  which  claim  general  efficacy  against 
bacteria,  the  standard  test  bacteria  should  be  tested.  For  these  products  efficacy  against 
Salmonella spp., Listeria spp. and Campylobacter jejuni is assumed, because they are more 
susceptible than the standard test bacteria. 

The  EN  standards  for  food  area  only  include  a  test  on  bacteriophages  but  not  for  other 
viruses. To demonstrate a general virus  claim a modified EN phase 2,  step 1 test (medical 
area  test  with  food  area  soiling)  can  be  provided  with  Adenovirus  and  Murine  Norovirus  as 
test organism and a DVG phase 2, step 2 test. 

When  the  product  is  intended  to  be  used  at  high  temperatures  (>40  ºC)  relevant  test 
organisms  for  these  temperatures  should  be  used  as  described  in  section  5.4.0.4.4  of  this 
Guidance. 

An overview of reference test organisms, also for high temperatures, is given in Appendix 3. 

Test conditions 

It  is  important  that  the  tests  are  performed  with  the  same  contact  time  as  claimed  on  the 
label.  The  claimed  contact  time  has  to  be  a  realistic  value.  For  manual  dishwashing 
disinfectants the contact time will be short (seconds), while industrial equipment disinfection 
by soaking in a solution can be very long (hours). 

In  general  dish  washing  disinfectants  should  be  tested  under  dirty  conditions,  since  these 
products  are  mainly  used  for  combined  cleaning  and  disinfection.  Tests  under  clean 
conditions will only suffice when the label instructions state that cleaning prior to disinfection 
is necessary. If this is not stated on the label the test should be done under dirty conditions. 

 
   
 
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Tests should be  carried out with soiling for  clean or dirty conditions in accordance with the 
test requirements. 

Note that for use in specific industries different types of soiling for dirty conditions should be 
used. The soiling needed for clean and dirty conditions can be found in the relevant EN tests 
or EN 14885 (version 2014 or later) and referenced in Appendix 4. 

The test temperature should be according to the use instructions on the label. 

Dish  washing  disinfectants  for  manual  use  are  normally  used  at  40ºC  and  therefore  tests 
should be done at this  temperature. When the product is used at lower temperatures (e.g. 
only for rinsing after normal dish washing with hot water) tests can be done at 20ºC. When 
the  intended  use  is  soaking,  starting  with  hot  water  and  after  which  the  solution  will  cool 
down during the contact time, this should also be taken into account in the tests. 

When disinfection is done at temperatures of 40 to 80 ºC the test temperature should be in 
accordance with the use and relevant test organisms should be used (see section 5.4.4.4.2 
of this Guidance). 

5.4.4.4.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and,  when 
applicable,  simulated-use  or  field  tests  have  been  performed  (using  the  required  test 
organisms and test conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT4 products 
the required log10 reductions tests are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

5.4.4.5 Disinfection in dish washing machines and crate washers 

5.4.4.5.1 Introduction 

Biocides  can  be  used  to  disinfect  dishes,  equipment,  crates,  boxes,  etc.  in  industrial  or 
dishwashing machines. 

This can be used in areas such as food or feed industry,  kitchens in restaurants or homes, 
shops like butchers and grocery shops were food is processed, etc. 

5.4.4.5.2 Data requirements 

Test methods 

For  efficacy  testing  of  equipment  and  dish  washing  disinfectants  the  tiered  approach  as 
described in section 5.4.0.4.1 of this Guidance is preferred. 

The following tests are normally required for these disinfectants: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a quantitative surface test (phase 2, step 2); 

•  and  simulated-use  or  field  test  (phase  3)  for  disinfectants  used  in  (dish)washing 

machines; 

all tests simulating practical conditions appropriate to its intended use (temperature, soiling, 
different surfaces, contact time, etc.). 

Several methods for testing the efficacy of hard surface disinfectants are available. 

 
 
 
 
 
   
 
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Appendices 2 and 4 give a list of recommended test methods. 

The  following  documents  are  recommended  for  surface  disinfection  in  dish  washing 
machines: 

•  EN 14885: gives an overview of which EN phase 2, step 1 and step 2 tests to use for 

different uses, 

The following test might be helpful for designing simulated-use or field tests: 

•  DIN SPEC 10534. 

Test organisms 

Equipment  and  dish  washing  disinfectants  should  be  at  least  sufficiently  effective  against 
bacteria and yeasts. Efficacy tests with these organisms should always be provided. 

For  uses  in  industrial  dish  washers  for  specific  purposes,  an  exception  can  be  made  when 
sound justification is provided. This will be evaluated on a case-by-case basis. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. Relevant groups of test organisms, next to bacteria and yeasts, can be 
fungi  (fungal  spores),  viruses,  bacteriophages,  and  bacterial  spores.  Bacteriophages  are 
mainly of importance in the dairy industry. 

The test organisms used in efficacy tests are normally stated in the applicable standard test 
methods.  For  dish  washing  disinfectants  Salmonella  spp.,  Listeria  spp.  and  Campylobacter 
jejuni  are  relevant  target  organisms.  For  products  which  claim  general  efficacy  against 
bacteria,  the  standard  test  bacteria  should  be  tested.  For  these  products  efficacy  against 
Salmonella spp., Listeria spp. and Campylobacter jejuni is assumed, because they are more 
susceptible than the standard test bacteria. 

The  EN  standards  for  food  area  only  include  a  test  on  bacteriophages  but  not  for  other 
viruses. To demonstrate a general virus  claim a modified EN phase 2,  step 1 test (medical 
area  test  with  food  area  soiling)  can  be  provided  with  Adenovirus  and  Murine  Norovirus  as 
test organism and a DVG phase 2, step 2 tests. 

When  the  product  is  intended  to  be  used  at  high  temperatures  (>40  ºC)  relevant  test 
organisms  for  these  temperatures  should  be  used  as  described  in  section  5.4.0.4.4  of  this 
Guidance. 

An overview of reference test organisms, also for high temperatures, is given in Appendix 3. 

Test conditions 

It  is  important  that  the  tests  are  performed  with  the  same  contact  time  as  claimed  on  the 
label. The claimed contact time has to be a realistic value. It will depend on the contact time 
for  the  disinfection  cycle  in  (dish)washing  machines.  Justification  for  the  used  contact  time 
should be given. 

In  general,  dish  washing  disinfectants  should  be  tested  under  dirty  conditions  since  these 
products  are  mainly  used  for  combined  cleaning  and  disinfection.  Tests  under  clean 
conditions will only suffice when the label instructions state that cleaning prior to disinfection 
is necessary or when this is incorporated in a previous cycle of the (dish)washing machine. 
If this is not stated on the label the test should be done under dirty conditions. 

Tests should be  carried out with soiling for  clean or dirty conditions in accordance with the 
test requirements. 

 
   
 
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Note that for use in specific industries different types of soiling for dirty conditions should be 
used. 

The soiling needed for clean and dirty conditions can be found in the relevant EN tests or EN 
14885 (version 2014 or later) and referenced in Appendix 4. 

For products intended to be added to (dish)washing machines, information on the following 
in-use conditions should be provided: 

• 

• 

• 

• 

the concentration of the product (or at least the active substance) in the water during 
disinfecting  process  (i.e.  washing  or  rinsing).  The  water  volume  used  can  differ 
between wash and rinse  cycle and different  washing programmes, but also  between 
dish washing machines; 

the water to dishes ratio in the test is an important factor that should reflect the in-
use conditions; 

the  temperature  during  the  disinfection  process  (high  when  added  in  wash  process, 
low in rinse process); 

the  contact  time  (differs  between  various  washing  programmes  and  washing 
machines). 

The  laboratory  tests  should  be  performed  under  these  conditions.  The  conditions  for 
effective disinfection can normally only be carried out in professional dish washing machines. 

If  the  exact  conditions  cannot  be  met,  for  example,  in  household  machines,  reasonable 
worst case conditions must be tested. 

Worst case conditions, e.g.: 

• 

• 

• 

• 

the lowest temperature; 

the highest volume of water (i.e. maximum dilution of the product); 

the shortest contact time; 

the maximum load of dishes (i.e. smallest water to dishes ratio). 

The test temperature should be according to the use instructions on the label. 

When  the  product  is  used  at  lower  temperatures  (e.g.  only  for  rinsing  after  normal  dish 
washing  with  hot  water)  tests  can  be  done  at  20ºC.  When  disinfection  is  done  at 
temperatures of 40 to 80 ºC the test temperature should be in accordance with the use and 
relevant test organisms should be used (see section 5.4.4.5.2. of this Guidance). 

5.4.4.5.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated-use  or  field  tests  have  been  performed  (using  the  required  test  organisms  and 
test conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT4 products 
the required log10 reductions tests are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  but  must  be  justified  in  the  application.  The 
CA  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting  the  other  CAs  where 
appropriate, and decide whether it is acceptable or not. 

 
 
 
 
 
   
 
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5.4.4.6 Disinfection of inner surfaces in human drinking water systems 

5.4.4.6.1 Introduction 

Biocides can be used to disinfect surfaces in human drinking water systems (further referred 
to as drinking water. This can be large water systems in drinking water companies, transport 
pipes  in  between  drinking  water  companies  (semi-finished  product),  the  communal  piping 
system, collective drinking water systems (hospitals and other health care facilities, hotels, 
penitentiary institutions, etc.),  and tanks and  reservoirs  for drinking water (for instance  on 
ships). 

When water systems are disinfected in closed circuits, after which the system is washed with 
clean water, it is considered to be disinfection of the pipework and is included in PT4. When 
disinfection  is  performed  in  water  systems  while  they  are  in  service  and  the  water  is  also 
disinfected the application is considered to be included in PT5. 

The drinking water systems may be new or rehabilitated drinking water pipes (e.g. in newly 
built  or  renovated  houses)  or  systems  that  are  in  service  for  some  time  and  have  become 
contaminated during this period. 

The  main  need  to  clean  and  disinfect  the  systems  is  to  get  a  fresh  start  of  the  system. 
Cleaning and disinfection programs may be combined to treat these systems. 

The  systems  that  have  been  in  service  for  some  time  contain  biofilm  and  organisms  to  be 
controlled might hide in this biofilm. For instance, Legionella can multiply in the biofilm. 

5.4.4.6.2 Data requirements 

Test methods 

For  efficacy  testing  of  biocidal  products  used  on  inner  surfaces  of  drinking  water  systems, 
the tiered approach as described in section 5.4.0.4.1 of this Guidance is preferred. 

For combined cleaning and disinfecting of drinking water pipes, the following test is normally 
required:  

•  a quantitative suspension test (phase 2, step 1). 

When efficacy against Legionella is claimed, the following tests are normally required: 

•  a quantitative suspension test (phase 2, step 1); 

•  and a field test (phase 3). 

all  simulating  practical  conditions  appropriate  to  its  intended  use  (temperature,  soiling, 
contact time, etc.). When ring trial validated test protocols for simulated-use tests (phase 2, 
step 2) become available these might replace the field trial. 

When efficacy against biofilms is claimed, the following tests are normally required: 

•  a quantitative suspension test (phase 2, step 1); 

•  a simulated-use test or a field test. 

Laboratory tests 

EN phase 2, step 1 tests for the food industrial, domestic and institutional area are relevant 
for this use. Efficacy against Legionella can be tested in EN 13623 (phase 2, step 1). 

See section 5.4.2.11 of this Guidance for biofilm test methods. 

Appendices 2 and 4 give a list of recommended test methods. 

 
   
 
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Field trials 

For  products  which  claim  efficacy  against  Legionella,  field  trials  with  the  following 
requirements should be provided: 

•  before testing it should be established that the installation contains high numbers of 
Legionella  (>100cfu/L).  A  zero-time  measurement  should  be  performed.  Systems 
must not be inoculated with micro-organisms in order to perform the efficacy test; 

•  a field trial should be performed in a system that has been in service for some time 

and has become infected during this period; 

• 

the  number  of  sampling  points  per  location  will  depend  on  the  number  of  draw-off 
points in the installation. The table below should be used; 

Table 7: Number of sampling points  

Number of draw-off points (outlets)  Number of sampling points 

10-100 

101 – 200 

201 – 400 

401 – 800 

801 – 1600 

> 1600 

4 

6 

8 

10 

12 

14 

* a draw-off point is a point where drinking water, household water or warm water is made 
available for use. 

•  after disinfection and subsequent washing of the system with clean water (removal of 
disinfectant),  samples  should  be  taken  and  the  amount  of  bacteria  (general)  and 
Legionella in the water should be determined. Samples should be taken 48 hours and 
2 weeks after disinfection; 

•  after  treatment,  water  from  none  of  the  sampling  points  should  contain  more  than 

100 colony forming units/litre Legionella. 

Test organisms 

Biocidal  products  for  drinking  water  disinfection  should  be  at  least  sufficiently  effective 
against  bacteria.  The  test  organisms  used  in  efficacy  tests  are  stated  in  the  applicable 
standard test methods. Efficacy tests with these organisms should always be provided. 

For products which claim efficacy against  Legionella, a test with Legionella spp. should also 
be performed. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. 

Test conditions 

It  is  important  that  the  tests  are  performed  with  the  same  contact  time  as  claimed  on  the 
label. The claimed contact time has to be a realistic value. 

Laboratory  phase  2,  step  1  tests  should  be  carried  out  with  soiling  for  clean  conditions  in 
accordance with the test requirements. The soiling needed for clean conditions can be found 

 
 
 
 
 
   
 
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in  the  relevant  EN  tests  and  referenced  in  Appendix  4.  Simulated-use  tests  should  be 
performed with relevant soiling. 

5.4.4.6.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory,  or  when 
applicable,  field  tests  have  been  performed  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where  pass  criteria  are  available  in  the  standard  test  these  should  be  met.  For  these 
products the required log10 reductions in the laboratory tests are referenced in Appendix 4. 
The field trial should not contain more than 100 colony forming units Legionella per litre. 

5.4.4.7 Disinfection of inner surfaces in veterinary water systems 

5.4.4.7.1 Introduction 

Biocides can be used to disinfect surfaces in veterinary water systems in farms, bio-industry, 
etc..  These  are  water  systems  provide  water  for  animals  to  drink,  to  prepare  feed,  and  to 
use for cleaning the area. Water systems that are also suitable for human drinking water are 
not included in this section (see previous section of this Guidance). 

When water systems are disinfected in closed circuits, after which the system is washed with 
clean water, it is considered to be disinfection of the pipework and is included in PT4. When 
disinfection  is  performed  in  water  systems  while  they  are  in  service  and  the  water  is  also 
disinfected the application is considered to be included in PT5. 

The  water  of  these  systems  can  be  provided  by  drinking  water  companies  but  can  also 
contain well, ground, or ditch water that is pumped up at the location, or other water. Water 
systems  in  livestock  farming  can  be  used  to  supply  food  additives  or  antibiotics  to  the 
animals.  Therefore,  these  veterinary  water  systems  may  be  more  fouled  than  human 
drinking water systems. 

5.4.4.7.2 Data requirements 

Test methods 

For  the  combined  cleaning  and  disinfecting  of  veterinary  drinking  water  pipes  (e.g.  water 
tanks, water in animal housings etc. used as drinking water for animals and for other uses in 
stables  like  cleaning,  preparing  feed,  etc.),  efficacy  should  be  demonstrated  in  a  tiered 
approach as described in section 5.4.0.4.1 of this Guidance. This includes a phase 2, step 1 
and step 2 test. 

The  following  documents  are  recommended  for  disinfecting  of  veterinary  drinking  water 
pipes: 

•  EN 14885 gives an overview of which EN phase 2, step 1 and step 2 tests to use for 
different uses, the tests (bactericidal) for the food area are relevant for this use; 

if  CEN  standards  are  not  relevant  or  available  for  the  use  or  organisms  claimed  the 
following documents are recommended if appropriately reflecting the application: 

•  OECD  guidance  for  the  testing  of  chemicals:  Quantitative  method  for  evaluating 
activity of microbiocides used on hard non-porous surfaces. (These are surface tests 
which would be considered phase 2, step 2 tests). 

The  use  of  the  specified  tests  is  strongly  recommended  where  they  are  relevant  and 
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When  efficacy  against  biofilms  is  claimed,  a  simulated-use  test  or  field  test  has  to  be 
performed, as well as a phase 2, step 1 test. See section 5.4.2.11 of this Guidance for test 
methods. 

Test organisms 

Biocidal  products  for  drinking  water  disinfection  should  be  at  least  sufficiently  effective 
against  bacteria.  The  test  organisms  used  in  efficacy  tests  are  stated  in  the  applicable 
standard test methods. Efficacy tests with these organisms should always be provided. 

For all other groups of organisms, data only have to be provided when activity against those 
organisms is claimed. 

Test conditions 

It  is  important  that  the  tests  are  performed  with  the  same  contact  time  as  claimed  on  the 
label. The claimed contact time has to be a realistic value. 

Laboratory tests should be carried out with soiling for clean or dirty conditions in accordance 
with  the  test  requirements  for  the  food  area.  Tests  under  clean  conditions  will  only  suffice 
when  the  label  instructions  state  that  cleaning  of  the  water  systems  prior  to  disinfection  is 
necessary. If this is not stated on the label the test should be done under dirty conditions. 

5.4.4.7.3 Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory,  or  when 
applicable,  simulated-use  or  field  tests  have  been  performed  (using  the  required  test 
organisms and test conditions), and when the pass criteria for the tests have been met. 

Where  pass  criteria  are  available  in  the  standard  test  these  should  be  met.  For  these 
products the required log10 reductions in the laboratory tests are referenced in Appendix 4. 

5.4.4.8 Other uses in PT4 

Several  uses  of  PT4  products  have  been  specified  in  the  above  sections  and  data 
requirements and  acceptance  criteria for these uses are described. For products with  other 
uses,  that  do  not  fit  in  one  of  the  described  uses,  it  is  up  to  the  applicant  to  demonstrate 
efficacy in an appropriate way. 

In  general  the  tiered  approach  as  described  in  section  5.4.0.4.1  of  this  Guidance  is 
preferred. Where possible the standard tests required for the described uses should be taken 
(e.g. EN phase 2, step 1 and step 2 tests for food area). Where the tests are not appropriate 
for the product, other tests can be used. In that case, a justification for the relevance of the 
tests used should be provided. The test design should be discussed with and agreed by the 
CA  before  testing  takes  place.  The  evaluation  will  be  done  on  a  case-by-case  basis  by  the 
CAs. 

The guidance will be updated when new methods become available. 

5.4.5 PT5 Drinking water disinfectants 

5.4.5.1 Introduction 

Product type 5 contains biocidal products used for the disinfection of drinking water for both 
humans  and  animals.  The  definition  of  drinking  water  is  in  accordance  with  Article  2  of 
Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human 
consumption. In this Guidance (and section) the term drinking water for humans is not only 

 
 
 
 
 
   
 
 
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used  for  water  that  will  be  consumed  directly  by  humans  but  also  for  other  uses  of  water 
coming out of the plumbing system like showering, cooking, etc. 

When  disinfection  is  done  in  the  water  system  while  it  is  in  service  and  the  water  itself  is 
also  disinfected,  this  is  included  in  PT5.  When  water  systems  are  disinfected  in  closed 
circuits,  after  which  the  system  is  washed  with  clean  water,  this  is  disinfection  of  the 
pipework only and is as such included in PT4. 

Disinfectant  products  can  be  added  to  drinking  water,  intermittently  by  shock  dosing  or 
continually dosing. The purpose of this type of disinfection is to disinfect the water in order 
to  prevent  transmission  of  water-borne  diseases  via  drinking  water.  Water-borne 
transmitted  pathogens  can  be  bacteria,  viruses,  yeasts,  fungal  spores  or  protozoan 
parasites. Disinfection is only one aspect of drinking water treatment. Application of drinking 
water  disinfectants  is  accompanied  with  the  responsibility  to  also  control  any  toxic 
disinfectant by-products. Treatment substances should only be added for specific hygienic or 
technical  reasons,  limiting  application  to  the  minimum  volumes  that  are  absolutely 
necessary  for  achieving  the  targeted  effect  (principle  of  minimisation)  and  only  under 
conditions optimising their efficacy. 

Disinfection within PT5 can be divided into six application groups: 

1.  Disinfection at the drinking water suppliers and their water distribution systems 

2.  Disinfection of raw water for individual supply (1-2 premises) 

3.  Disinfection in collective drinking water systems 

4.  Disinfection of water in reservoirs 

5.  Disinfection  of  water  of  undefined  quality  for  small  scale  use  (up  to  5 

L/person/day) 

6.  Disinfection of water for animals 

In the sections below a detailed description  of  each group as well as the requirements and 
acceptance criteria for most common uses are specified. For other uses and claims that are 
not specifically mentioned the requirements will be set on a case-by-case basis by the CAs. 

5.4.5.2 Disinfection  at  the  drinking  water  suppliers  and  their  water  distribution 
systems 

5.4.5.2.1 Introduction 

This is the disinfection of water during drinking water treatment in water plants of drinking 
water  suppliers,  during  transport  in  between  drinking  water  suppliers,  and  prior  to 
distribution into (part of) the communal piping system (referred to as primary disinfection in 
this guidance). This group also includes products that are added by drinking water suppliers 
to the previously-treated water already in the  public distribution network to ensure that an 
adequate  disinfectant  residual  is  maintained  throughout  the  system  (referred  to  as 
secondary disinfection in this guidance). 

Following  physical  treatment  of  water,  primary  disinfection  describes  the  main  disinfection 
method  employed 
inactivate  waterborne  pathogenic  micro-organisms.  Primary 
disinfection  is  often  supplemented  by  downstream  secondary  disinfection  to  maintain  a 
residual level of disinfectant within the distribution system in order to assure good quality of 
drinking  water  to  the  point  of  compliance  i.e.  the  consumer’s  tap  as  determined  in  the 
Drinking Water Directive. 

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5.4.5.2.2 Data requirements 

Test methods 

For  product  authorisation  of  drinking  water  disinfectants  used  by  the  drinking  water 
suppliers  and  in  water  distribution  systems,  the  tiered  approach  as  described  in  section 
5.4.0.4.1 of this Guidance is preferred. 

Next  to  a  suspension  test,  a  simulated  use  test  should  be  performed.  For  suspension  tests 
EN phase 2, step 1 tests are preferred. Since for most target organisms there are no specific 
EN tests for drinking water disinfection, tests should be modified to reflect the use conditions 
with  respect  to  soiling,  temperature  range  and  contact  time.  EN  tests  from  food  and 
industrial  area  (see  EN  14885)  can  be  modified  (see  ‘Test  conditions’  on  next  page).  For 
virucidal activity EN 14476 can be modified. 

For  the  simulated  use  test  for  primary  disinfection  a  detailed  appropriate  test  method  is 
given  in  the  test  method  “Quantitative  determination  of  the  efficacy  of  drinking  water 
disinfectants”  (see  Appendix  2  Table  32).  The  test  is  performed  on  an  adapted  test  rig.  A 
disinfectant  neutralizer  or  filter  system  is  required  to  stop  a  reaction  between  disinfectant 
and test organisms. Currently the simulated-use test can only be performed in the test lab in 
Germany where the test was developed, as only there the required test  set up is available. 
Alternative  methods  will  be  considered  and  are  acceptable  provided  they  are  scientifically 
justified  and  will  be  evaluated  by  the  CA  on  a  case-by-case  basis.  Please  note  that 
monitoring data can only be accepted as supplementary data since this data does not offer 
the possibility to calculate log reduction to evaluate the disinfection. 

For secondary disinfection a simulated-use test is required with relevant use conditions with 
respect to temperature, soiling and contact time. 

For an overview of available EN tests see Appendices 2 and 4. 

Test organisms 

Drinking  water  disinfectants  used  on  site  at  the  drinking  water  suppliers  and  water 
distribution systems should be at least sufficiently effective against bacteria and viruses. 

Efficacy  tests  with  these  organisms  should  always  be  provided.  For  all  other  groups  of 
organisms  (Protozoa,  etc.),  data  only  have  to  be  provided  when  activity  against  those 
organisms  is  claimed.  The  test  organisms  used  in  efficacy  tests  are  normally  stated  in  the 
applicable  standard  test  methods  or  the  test  method  “Quantitative  determination  of  the 
efficacy  of  drinking  water  disinfectants”.  For  drinking  water  disinfectants  used  on  site  at 
drinking  water  suppliers  and  water  distribution  systems  Staphylococcus  aureus, 
Pseudomonas aeruginosa, Enterococcus hirae and Escherichia coli should be tested. 

In an EN suspension test the efficacy against enteroviruses and norovirus should be tested. 
In  the  simulated  use  test  bacteriophages  are  used  as  an  indicator  for  human  viruses  as 
given  in  the  test  method  “Quantitative  determination  of  the  efficacy  of  drinking  water 
disinfectants”. 

An overview of reference test organisms is given in Appendix 3. 

Test conditions 

It is important that the efficacy tests are carried out with the contact time as claimed on the 
label, and also that the claimed contact time is a realistic value. 

Suspension  tests  may  be  modified  considering  the  type  of  disinfectant  application  and 
considering  especially  that  the  test  needs  to  be  performed  reflecting  the  worst-case 

 
 
 
 
 
   
 
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conditions  (temperature,  soiling,  contact  time,  mineralization,  pH).  Further  details  can  be 
taken from Appendix 4. For suspension tests the maximum contact time is 30 minutes. For 
simulated use tests contact time of 10 and 25 minutes should be applied. 

Laboratory  tests  should  be  carried  out  with  appropriate  soiling.  For  primary  disinfection  it 
can  be  expected  that  soiled  water  is  used  e.g.  surface  water.  Therefore,  for  this  use  the 
laboratory  tests  should  be  done  under  dirty  conditions.  Secondary  disinfection  is  done  on 
clean water, simulated by clean test conditions. Appendix 4 states the appropriate soiling for 
PT5. 

The applicant should provide the rational for the choices made. 

Acceptance criteria  

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated  use  tests  have  been  carried  out  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT5 products 
the required log reductions in suspension tests are referenced in Appendix 4. 

The  pass  criteria  for  the  simulated-use  test  are  stated  in  the  test  (see  Appendix  4).  The 
same criteria are valid for both primary and secondary disinfection. 

Deviations from the pass criteria are possible, but must be justified in the application. If the 
simulated  use  test  passed  but  the  suspension  test  did  not  pass,  the  applicant  needs  to 
justify  why  the  concentration  used  in  the  simulated  use  test  should  be  considered  as  the 
effective dose. 

The  Competent  Authority  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting 
the  other  Competent  Authorities  where  appropriate,  and  decide  whether  it  is  acceptable  or 
not. 

5.4.5.3 Disinfection of raw water for individual supply (1-2 premises) 

5.4.5.3.1 Introduction 

These are disinfectants intended to be used for private water supply, (i.e. any water supply 
which  is  supplied  to  a  property  that  is  not  provided  by  a  water  supplier).  Most  of  these 
supplies  are  situated  in  remote,  rural  parts  of  a  country  and  can  originate  from  a  range  of 
sources including wells, natural springs and watercourses. 

5.4.5.3.2 Data requirements 

Test methods 

For  product  authorisation  of  drinking  water  of  individual  supply  the  tiered  approach  as 
described in section 5.4.0.4.1 of this Guidance is preferred. 

Next to a suspension test a simulated use test should be performed. For suspension tests EN 
phase 2, step 1 tests are preferred. Since for most target organisms there are no specific EN 
tests  for  drinking  water  disinfection,  tests  should  be  modified  to  reflect  the  use  conditions 
with  respect  to  temperature  range,  soiling  and  contact  time.  EN  tests  from  food  and 
industrial  area  (see  EN  14885)  can  be  modified  (see  ‘Test  conditions’  on  next  page).  For 
virucidal activity EN 14476 can be modified. 

For  the  simulated  use  test,  a  detailed  appropriate  test  method  is  given  in  the  test  method 
“Quantitative  determination  of  the  efficacy  of  drinking  water  disinfectants”.  The  test  is 
performed  on  an  adapted  test  rig.  A  disinfectant  neutralizer  or  filter  system  to  stop  a 

 
   
 
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reaction  between  disinfectant  and  test  organisms  is  required.  Currently  the  simulated-use 
test  can  only  be  performed  in  Germany.  Alternative  methods  will  be  considered  and  are 
acceptable  provided  they  are  scientifically  justified  and  will  be  evaluated  by  the  CA  on  a 
case-by-case basis. 

For an overview of available EN tests see Appendices 2 and 4. 

Test organisms 

Drinking water disinfectants of raw water for individual supply should be at least sufficiently 
effective against bacteria and viruses. Efficacy tests with these organisms should always be 
provided. For all other groups of organisms (Protozoa, etc.), data only have to be provided 
when activity against those organisms is claimed. 

The test organisms used in efficacy tests are normally stated in the applicable standard test 
methods  or  the  test  method  “Quantitative  determination  of  the  efficacy  of  drinking  water 
disinfectants”. For drinking water disinfectants used in private drinking water supply systems 
Staphylococcus  aureus,  Pseudomonas  aeruginosa,  Enterococcus  hirae  and  Escherichia  coli 
should be tested. 

In EN suspension tests efficacy against enteroviruses and norovirus should be tested. In the 
simulated  use  test,  bacteriophages  are  used  as  an  indicator  for  human  viruses  as  given  in 
the test method “Quantitative determination of the efficacy of drinking water disinfectants”. 

An overview of reference test organisms is given in Appendix 3. 

Test conditions 

It is important that the efficacy tests are carried out with the contact time as claimed on the 
label, and also that the claimed contact time is a realistic value.  

Suspension  tests  may  be  modified  considering  the  type  of  disinfectant  application  and 
considering  especially  that  the  test  needs  to  be  performed  reflecting  the  worst-case 
conditions  (temperature,  soiling,  contact  time,  mineralization,  pH).  Further  details  can  be 
taken from Appendix 4. For suspension tests the maximum contact time is 30 minutes. For 
simulated use tests contact time of 10 and 25 minutes should be applied. 

Laboratory tests should be carried out with soiling for dirty conditions as defined in Appendix 
4.  Depending  on  the  water  source  interfering  substances  may  be  variable  and  require 
modifications of the soiling in the efficacy tests. The applicant should provide the rational for 
the choices made. 

Further details can be taken from Appendix 4. 

Acceptance criteria  

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated  use  tests  have  been  carried  out  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT5 products 
the required log reductions in suspension tests are referenced in Appendix 4. 

The pass criteria for the simulated-use test are stated in the test. 

Deviations from the pass criteria are possible, but must be justified in the application. If the 
simulated  use  test  passed  but  the  suspension  test  did  not  pass,  the  applicant  needs  to 
justify  why  the  concentration  used  in  the  simulated  use  tests  should  be  considered  as  the 
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The  Competent  Authority  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting 
the  other  Competent  Authorities  where  appropriate,  and  decide  whether  it  is  acceptable  or 
not. 

5.4.5.4 Disinfection in collective drinking water systems 

5.4.5.4.1 Introduction 

This  is  disinfection  in  collective  drinking  water  systems  like  hospitals  and  other  health  care 
facilities, hotels, penitentiary institutions, etc. In these large plumbing systems water might 
become  contaminated  with  Legionella  spp.  In  addition  to  physical  techniques  (heating,  UV 
treatment, etc.) chemical disinfection is sometimes allowed in some EU countries. 

5.4.5.4.2 Data requirements 

Test methods 

For product authorisation of drinking water disinfectants in collective drinking water systems 
the tiered approach as described in section 5.4.0.4.1 of this Guidance is preferred. 

The  following  requirements  are  set  for  biocides  to  be  used  as  disinfectant  in  collective 
drinking water systems: 

Laboratory tests 

Basic efficacy of the product should be demonstrated in suspension tests (phase 2, step 1). 

Studies  should  show  that  the  product  can  accomplish  a  log  reduction  of  5  against  bacteria 
and  a  log  reduction  of  4  against  Legionella  pneumophila  specifically.  This  can  be  done  in 
laboratory tests (e.g. suspension tests EN 1276 and EN 13623). Tests should be modified to 
reflect  the  use  conditions  with  respect  to  soiling,  temperature  range  and  contact  time  (see 
Appendix 4). 

The suspension tests can be waived when simulated use or field trials are available in which 
the  concentration  of  Legionella  spp.  is  high  enough  to  show  log  reduction  of  5  (min.  105 
cfu/L). 

Simulated use tests 

A  simulated  use  test  should  be  performed  but  is  only  mandatory  in  cases  where  a  log 
reduction of 4 cannot be demonstrated in a field trial due to low levels of  Legionella spp. in 
the drinking water or in the suspension test. 

A  detailed  description  for  simulated  use  test  is  given  in  the  test  method  “Quantitative 
determination of the efficacy of drinking water disinfectants. Currently this test can only be 
performed in Germany. Alternative methods will be considered and are acceptable provided 
they are scientifically justified: they will be evaluated by the CA on a case-by-case basis. If 
this  test  cannot  be  used  according  to  the  scope  of  the  test  an  alternative  method  can  be 
presented.  CAs  will  examine  the  eligibility  of  the  proposed  alternative.  As  the  test  method 
“Quantitative  determination  of  the  efficacy  of  drinking  water  disinfectants”  does  not  cover 
Legionella spp., an experimental method to simulate a system with hot water is given in the 
following publications: “Development of a pilot-scale 1 for Legionella elimination in biofilm in 
hot  water  network:  heat  shock  treatment  evaluation”  and  “Chemical  disinfection  of 
Legionella in hot water system biofilm: A pilot-scale 1 study” (see Appendix 2, Table 32). 

 
   
 
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Field trials 

Field trials (historic and in use monitoring) should always be provided especially for products 
with  long  and  continuous  use.  See  below  under  Test  Conditions/Field  Trials  for  further 
details. 

Test organisms 

PT5  products  for  collective  drinking  water  systems  should  be  at  least  sufficiently  effective 
against bacteria and specifically against  Legionella  spp. Since the control  of  Legionella spp. 
in collective drinking water systems is of major importance, efficacy against  Legionella spp. 
(field  tests)  and  Legionella  pneumophila  (suspension  tests  or  simulated  use  tests)  should 
always be demonstrated in addition to general test against bacteria. 

Efficacy tests with these organisms should always be provided. 

For  all  other  groups  of  organisms,  data  only  need  to  be  provided  when  an  efficacy  against 
those organisms is claimed. 

Test conditions 

Laboratory tests 

It is important that the efficacy tests are carried out with the contact time as claimed on the 
label, and also that the claimed contact time is a realistic value. 

Suspension  tests  may  be  modified  considering  the  type  of  disinfectant  application  and 
considering  especially  that  the  test  needs  to  be  performed  reflecting  the  worst-case 
conditions  (temperature,  soiling,  mineralization,  pH).  Further  details  can  be  taken  from 
Appendix 4. For suspension tests the maximum contact time is 24 hours. 

Since  the  water  treated  in  collective  drinking  water  system  is  clean  water  coming  from  a 
drinking company, laboratory tests should be carried out with soiling for clean conditions as 
defined in Appendix 4. 

Simulated use tests 

The tests are carried out with the standard contact time (10 and 25 minutes) or as claimed 
on  the  label.  Tests  should  be  carried  out  with  soiling  for  clean  conditions  as  defined  in 
Appendix 4. 

Field Trials 

LOCATIONS 

A field trial should be performed at a minimum of 3 locations. 

The  drinking  water  quality  in  the  different  EU  countries  may  differ.  In  some  EU  countries 
disinfectants like chlorine are included as standard, whilst in other countries disinfectants are 
only  added  during  outbreaks  of  pathogens.  Therefore  some  EU  countries  will  only  accept 
field  trials  carried  out  within  their  own  country  or  in  locations  with  comparable  water 
specifications. In general, however, tests are not performed in all EU countries. Therefore, in 
all  field  tests  the  quality  of  the  tested  drinking  water  should  be  clearly  specified  and 
documented. The comparability of this water to the drinking water in each country should be 
clearly described and justified, accordingly. Ultimately, the Competent Authority will decide 
whether the test is acceptable or not. 

Only locations with 100 or more operational draw-off points (downstream of the application 
spot) are acceptable. A location is a collective drinking water system which is treated by the 

 
 
 
 
 
   
 
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product. Also a part of a collective drinking water system, for instance a wing of a building 
or only the cold water system, can be seen a test location as long as it contains 100 or more 
operational draw-off points. 

DURATION OF THE TEST 

When  the  apparatus  is  in  continuous  or  discontinuous  use  (so  no  single  applications)  the 
duration  of  the  test  is  one  year  per  location,  starting  from  the  first  sampling  round  after 
starting the apparatus. When, due to starting problems etc., the first months do not give the 
required result, the test should be extended to ensure a duration of one year starting from 
the point at which a stable situation is reached. In this way at least a year of test results can 
show that the product is capable of controlling Legionella spp. 

DIFFERENT TYPES OF WATER 

It is recommended that the locations are spread over the country, this is to ensure that the 
product  is  tested  on  different  types  of  water  (hardness,  organic  material,  etc.).  For  this 
purpose information should be provided on the quality of the provided water at the different 
locations. In principal this information is available through the water suppliers. 

LEGIONELLA 

Before  starting  a  test  it  should  be  clear  that  the  installation  to  be  treated  is  contaminated 
with Legionella spp. bacteria (≥1000 cfu/L). For this purpose information should be provided 
on  (recent)  problems  with  Legionella  spp.,  like  results  from  sampling  in  the  past  and 
performed cleanings, etc. The system should not be artificially contaminated. 

SAMPLING POINTS 

The amount of sampling points per location depends on the amount of draw-off points (taps 
and other outlets) in the installation. The table below should be used. 

Table 8: Number of sampling points 

Number of draw-off points (outlets)  Number of sampling points 

101 – 200 

201 – 400 

401 – 800 

801 – 1600 

> 1600 

6 

8 

10 

12 

14 

All sampling points should be unambiguously coded. 

At  each  sampling  round  two  sampling  points  are  sampled  each  time  (standard  sampling 
points),  preferably  the  sampling  point  next  to  the  apparatus  and  the  sampling  point  the 
most  far  away  from  it.  These  sampling  points  should  be  clearly  described  and  the  code  of 
these points should be  stated. All  other  sampling points may vary at  each sampling round. 
When  a  sampling  point  shows  elevated  values  of  Legionella  spp.  or  one  of  the  other 
parameters this sampling point should be sampled again the next month. The total amount 
of sampling points should remain the same, according to the table above. 

 
   
 
 
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The tuning of the apparatus from which the disinfectant is dosed should be recorded at  the 
time of sampling. 

EFFICACY 

The following measurements should be performed: 

• 

zero  measurement:  measurement  of  Legionella  spp.,  total  hardness,  pH,  organic 
contamination  of  the  water  and  residues  of  active  substances  from  previous 
treatments before the disinfection treatment is started. 

•  Legionella  spp.,  monthly  sampling,  norm  value  100  cfu/l  (90%-percentile  with  a 

maximum of 1000 cfu/l); 

• 

total hardness, Ca, Mg; sampling once per four months, depending on the variation a 
higher frequency might be necessary; also data from the water supplying companies 
can be collected; 

•  pH,  monthly  sampling  on  both  standard  sampling  points,  or  data  from  the  water 

supplying companies can be collected. 

ACTIVE SUBSTANCES  

To  determine  the  amount  of  active  substance  in  the  water,  the  relevant  substances  should 
be measured monthly. 

In  general  the  active  substance  of  the  used  biocidal  product  should  be  measured  monthly. 
The sampling point as  stated in Table 8  of this section should be taken. Especially the first 
and  the  most  far  away  sampling  point  are  of  importance,  in  order  to  ensure  that  enough 
product  reaches  the  end  of  the  system.  These  data  are  especially  relevant  for  the  efficacy 
assessment  of  in  situ  generated  products  and  can  also  be  used  in  other  areas  (e.g.  for 
toxicological and environmental risk assessment). 

GENERAL REQUIREMENTS FOR STUDY REPORTS 

Every  study  report  should  contain  a  good  description  of  the  material  (location,  number  of 
draw-off  points,  sampling  points,  history  of  Legionella,  etc.),  the  method  (starting  date, 
tuning  of  the  apparatus  from  which  the  disinfectant  is  dosed)  and  the  results  (including  0-
measurement).  In  the  study  reports  of  the  field  tests  the  results  should  be  interpreted  per 
location. Remarks such as high values above the norm, should be mentioned and explained. 
The report should contain a conclusion. 

APPARATUS 

In case  of in  situ production of the active substance  or  when an apparatus is used to dose 
the active substance in the right amount to the water, the report should contain information 
on safety measurements concerning over and under dosing. Continuous measurement of the 
dosed  active  substance  should  be  established.  The  devices  used  to  generate  the  active 
substance in situ themselves are not covered by the provision of BPR and consequently  are 
not subject to the authorisation. 

Acceptance criteria  

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory,  simulated 
use  and  field  tests  have  been  carried  out  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT5 products 
the required log reductions in suspension tests are referenced in Appendix 4. 

 
 
 
 
 
   
 
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The pass criteria for the simulated-use test are stated in the test (see Appendix 4). 

For the evaluation of the results of the measurements in the field trial, the norm values used 
are  mentioned  above  under  Test  Conditions/Field  Trials.  Per  location,  90%  of  the 
measurements  should  fulfil  the  requirements.  Over  all  locations  together,  90%  of  the 
locations should fulfil the requirements. 

Deviations  from  the  pass  criteria  are  possible,  however  they  must  be  justified  in  the 
application. 

The  Competent  Authority  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting 
the  other  Competent  Authorities  where  appropriate,  and  decide  whether  it  is  acceptable  or 
not. 

5.4.5.5 Disinfection of water in reservoirs 

5.4.5.5.1 Introduction 

This  is  disinfection  of  water  stored  in  tanks  and  reservoirs,  for  instance  on  ships,  mobile 
homes, or in small tanks as in a dentist’s chair. It is presumed that these tanks start filled 
with water of drinking water quality. The disinfection product should maintain the quality of 
the water over time. When the product is also intended to disinfect water from other sources 
(e.g. ground water, spring or surface water) this should be clear in the claim for the product. 
It  should  also  be  specified  whether  the  tank  should  be  cleaned  before  disinfection  or  not. 
The claimed use should be specified in the SPC. 

5.4.5.5.2 Data requirements 

Test methods 

For product authorisation of drinking water disinfectants in reservoirs the tiered approach as 
described  in  section  5.4.0.4.1  of  this  Guidance  is  preferred.  Next  to  a  suspension  test  a 
simulated use test should be performed. 

For suspension tests EN phase 2, step 1 tests are preferred. Since for most target organisms 
there  are  no  specific  EN  tests  for  drinking  water  disinfection,  tests  should  be  modified  to 
reflect  the  use  conditions  with  respect  to  soiling,  temperature  range  and  contact  time.  EN 
tests  from  food  and  industrial  area  (see  EN  14885)  can  be  modified  (see  ‘Test  conditions’ 
below). For virucidal activity EN 14476 can be modified. Efficacy suspension tests should be 
provided  with  two  concentrations:  the  concentration  of  the  product  as  dosed  (start 
concentration) and the active substance concentration obtained in the field at the end of the 
claimed period of use. 

For disinfection of water in reservoirs it is mandatory to provide a simulated-use test. Such 
tests  are  required  in  order  to  demonstrate  proper  distribution  of  the  disinfectant  in  the 
reservoir.  In  the  absence  of  a  standard  method,  the  applicant  should  provide  a  testing 
proposal  which  needs  to  be  agreed  by  the  eCA  in  advance.  Alternatively,  for  products  with 
one  active  substance  that  can  easily  be  measured,  efficacy  can  be  demonstrated  using  a 
field test in which the amount of active substance and the amount of organisms is measured 
several times during the test period. 

In  some  cases  efficacy  against  biofilm  is  of  importance  in  this  use.  For  testing  efficacy 
against biofilms see section 5.4.2.11 of this Guidance. 

In cases when water is of drinking water quality in the beginning and the disinfectant is used 
to  maintain  water  quality,  information  should  be  provided  on  how  long  the  effect  can  be 

 
   
 
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guaranteed  at  a  certain  temperature  and  a  maximum  DOC.  This  should  be  justified  and 
demonstrated in the efficacy tests. 

For an overview of available EN tests see Appendix 2. 

Test organisms 

Drinking  water  disinfectants  for  reservoir  water  should  be  at  least  sufficiently  effective 
against bacteria and viruses. Tests with these organisms should always be provided. 

For  all  other  groups  of  organisms  (e.g.  Legionella  spp.  or  Protozoa)  tests  only  have  to  be 
provided when efficacy against these organisms are claimed. 

The test organisms used in efficacy tests are normally stated in the applicable standard test 
methods. An overview of reference test organisms for PT5 is given in Appendix 3. 

Test conditions 

It is important that the tests are carried out with the same contact time as claimed on the 
label.  The  claimed  contact  time  has  to  be  a  realistic  value.  Therefore,  the  applicant  has  to 
clearly  indicate  how  long  the  disinfectant  can  guarantee  the  quality  of  the  water  in  the 
reservoir.  When  started  with  raw  water  it  should  be  indicated  at  what  time  after  the 
treatment the water can be used. 

If protozoa are claimed, tests with longer contact times relevant for protozoa are acceptable. 

When  starting  with  water  of  drinking  water  quality,  tests  should  be  carried  out  with  soiling 
for clean conditions as stated in Appendix 4. For this type of product (if tested under clean 
conditions),  the  applicant  needs  to  clearly  indicate  to  the  user  that  the  reservoir  should  be 
clean before filling it with fresh and clean water. 

When starting with raw water, tests should be carried out with soiling for dirty conditions in 
accordance with the test requirements (see Appendix 4). 

Acceptance criteria  

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and 
simulated  use  tests  have  been  carried  out  (using  the  required  test  organisms  and  test 
conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. The required log 
reductions in suspension tests are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  however  they  must  be  justified  in  the 
application. 

The  Competent  Authority  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting 
the  other  Competent  Authorities  where  appropriate,  and  decide  whether  it  is  acceptable  or 
not. 

5.4.5.6 Disinfection  of  water  of  undefined  quality  for  small  scale  use  (up  to  5 
L/person/day) 

5.4.5.6.1 Introduction 

This  is  disinfection  of  for  instance,  individual  emergency  water  supply  or  other  water  that 
might  be  contaminated  in  places  where  no  clean  drinking  water  is  available.  This  means 
water not originally coming from the drinking water suppliers. This is only intended for water 
that is used directly for drinking or preparing food after disinfection, therefore for small scale 
use (up to 5 L/person/day). 

 
 
 
 
 
   
 
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5.4.5.6.2 Data requirements 

Test methods 

For this use it is in most cases acceptable to demonstrate efficacy in a suspension test only. 
For suspension tests EN phase 2, step 1 tests are preferred. Since for most target organisms 
there  are  no  specific  EN  tests  for  drinking  water  disinfection,  tests  should  be  modified  to 
reflect  the  use  conditions  with  respect  to  soiling,  temperature  range  and  contact  time.  EN 
tests from food and industrial area (see EN 14885) can be modified (see ‘Test conditions’ on 
the next page). For virucidal activity EN 14476 can be modified. For an overview of available 
EN tests see Appendix 2. 

If  due  to  turbidity  a  pre-treatment  is  needed,  such  as  filtration,  this  should  be  part  of  the 
test  conditions.  It  is  the  responsibility  of  the  applicant  to  clearly  instruct  that  a  pre-
treatment  is  required  due  to  turbidity.  This  should  also  be  reflected  on  the  SPC  of  the 
product in the section “Instructions of use” together with the exact treatment duration.  

If  no  pre-treatment  for  turbidity  is  involved  for  turbid  water,  the  field  trials  should  be 
performed.  Field  trials  should  be  performed  with  different  raw  water  (mineralisation,  TOC, 
temperature, pH) in which turbidity is considered. 

Test organisms 

Drinking water disinfectants of “water with undefined quality (small scale use)” should be at 
least  sufficiently  effective  against  bacteria  and  viruses.  For  all  other  groups  of  organisms 
tests  only  have  to  be  provided  when  efficacy  against  the  organisms  that  are  claimed.  The 
test  organisms  used  in  efficacy  tests  are  normally  stated  in  the  applicable  standard  test 
methods. An overview of reference test organisms is given in Appendix 3. 

Test conditions 

A  suspension  test  needs  to  be  performed  reflecting  worst-case  conditions  (temperature, 
soiling, contact time, mineralization, pH). The test should be done with the claimed contact 
time but no longer than 30 minutes.  The suspension test (EN phase 2, step 1  – food area) 
should be carried out with soiling for dirty conditions (see Appendix 4). 

For  the  field  trial  at  least  three  types  of  raw  water  should  be  tested.  Information  on 
mineralisation, TOC, temperature, pH and turbidity should be given. 

Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  and  field 
tests  have  been  carried  out  (using  the  required  test  organisms  and  test  conditions),  and 
when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT5 products 
the required log reductions in suspension tests are referenced in Appendix 4. 

Deviations  from  the  pass  criteria  are  possible,  however  they  must  be  justified  in  the 
application. 

The  Competent  Authority  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting 
the  other  Competent  Authorities  where  appropriate,  and  decide  whether  it  is  acceptable  or 
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5.4.5.7 Disinfection of water for animals 

5.4.5.7.1 Introduction 

This  is  disinfection  of  water  in  animal  housing  used  as  drinking  water  for  animals  and  for 
other  uses  in  animal  houses  (preparing  feed,  etc.).  When  products  are  used  to  disinfect 
water  for  both  humans  and  animals,  requirements  according  to  sections  5.4.5.2  to  5.4.5.4 
are  also  applicable.  The  origin  of  the  water  in  water  systems  for  animals  can  differ,  e.g. 
groundwater,  surface  water  (dirty),  or  water  from  drinking  water  suppliers  (clean).  The 
intended use should be specified on the SPC. 

5.4.5.7.2 Data requirements 

Test methods 

For efficacy testing of disinfectants for water for animals the tiered approach as described in 
section 5.4.0.4.1  of this Guidance is preferred. Next to a  suspension  test also a  simulated-
use  test  or  field  test  (phase  3)  should  be  performed,  to  provide  information  under  in-use 
conditions.  In  some  cases  efficacy  against  biofilm  is  of  importance  in  this  use.  For  testing 
efficacy  against  biofilms  see  section  5.4.2.11  of  this  Guidance.  For  suspension  tests  EN 
phase 2, step 1 tests are preferred. Since for most target organisms there are no specific EN 
tests  for  drinking  water  disinfection,  tests  should  be  modified  to  reflect  the  use  conditions 
with  respect  to  soiling,  temperature  range  and  contact  time.  EN  tests  from  food  and 
industrial  area  (see  EN  14885)  can  be  modified  (see  ‘Test  conditions’  on  next  page).  For 
virucidal activity EN 14476 can be modified. 

For  the  simulated  use  test  a  detailed  appropriate  test  method  is  given  in  the  test  method 
“Quantitative  determination  of  the  efficacy  of  drinking  water  disinfectants”.  The  test  is 
realised on an adapted test rig. A disinfectant neutralizer or filter system to stop a reaction 
between  disinfectant  and  test  organisms  is  required.  Currently  this  test  can  only  be 
performed in Germany. Alternative methods will be considered and are acceptable provided 
they are scientifically justified and will be evaluated by the CA on a case-by-case basis. 

Since  drinking  water  for  animals  can  be  obtained  from  a  variety  of  different  sources,  e.g. 
surface water (lakes, rivers), underground water pumped from wells, human drinking water, 
rain water, etc., several kinds of water should be tested. Alternatively, it should be indicated 
on the label under which conditions the product can be used. 

Test organisms 

Drinking  water  disinfectants  of  water  for  animals  should  be  at  least  sufficiently  effective 
against  bacteria.  For  all  other  groups  of  organisms  tests  only  have  to  be  provided  when 
efficacy  against  the  organisms  are  claimed.  The  test  organisms  used  in  efficacy  tests  are 
normally  stated  in  the  applicable  standard  test  methods.  An  overview  of  reference  test 
organisms is given in Appendix 3. 

Test conditions 

It is important that the efficacy tests are carried out with the contact time as claimed on the 
label, and also that the claimed contact time is a realistic value. 

Suspension  tests  may  be  modified  considering  the  type  of  disinfectant  application  and 
considering  especially  that  the  test  needs  to  be  performed  reflecting  the  worst-case 
conditions  (temperature,  soiling,  contact  time,  mineralization,  pH).  Further  details  can  be 
taken from Appendix 4. 

 
 
 
 
 
   
 
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Laboratory tests should be carried out with soiling for clean or dirty conditions as defined in 
Appendix  4.  Depending  on  the  water  source  that  has  to  be  disinfected  the  test  should  be 
performed under either clean or dirty (e.g. undefined or pumped up water) conditions. 

Field tests should be done in animal housing. A testing proposal needs to be provided taking 
into  consideration  relevant  parameters,  such  as  type  of  water  to  be  treated  (e.g.  water 
originating  from  the  public  distribution  system  or  surface  water),  pre-cleaning  of  the 
“distribution system”, pre-treatment of the water (e.g. physical treatment such as filtration) 
and application of food additives or antibiotics which will be evaluated by the CA on a case-
by-case basis. 

Acceptance criteria 

A  product  will  be  assessed  to  be  sufficiently  effective  if  the  required  laboratory  tests,  or 
when  applicable,  field  tests  have  been  carried  out  (using  the  required  test  organisms  and 
test conditions), and when the pass criteria for the tests have been met. 

Where pass criteria are available in the standard test these should be met. For PT5 products 
the required log reductions in suspension tests are referenced in Appendix 4. 

The pass criteria for the simulated-use test are stated in the test (see Appendix 2 Table 32). 
Field trials should demonstrate sufficient efficacy and the microbiological burden should stay 
below  an  acceptable  level  according  to  the  relevant  legislation.  Deviations  from  the  pass 
criteria are possible, however they must be justified in the application. 

The  Competent  Authority  will  evaluate  any  justification  on  a  case-by-case  basis,  consulting 
the  other  Competent  Authorities  where  appropriate,  and  decide  whether  it  is  acceptable  or 
not. 

5.4.6 Materials and Articles Treated to Protect Humans or Animals 

For  testing  materials  and  articles  with  claims  to  protect  humans  or  animals,  a  tailored 
approach is compulsory. The testing strategy entirely depends on the specific claim made. In 
the  majority  of  cases,  a  claim  can  only  be  made  for  a  specific  type  of  final  article,  as  use 
area  and  use  conditions  are  decisive  for  describing  the  problem  which  the  biocide  must 
solve,  and  to  demonstrate  efficacy  in  exactly  those  conditions  is  necessary.  Consequently, 
this  section  describes  testing  principles  and  strategies  rather  than  recommending  specific 
tests. 

A  tiered  approach  has  to  be  followed  in  demonstrating  claims  for  protection  of  humans  or 
animals: 

•  Tier  1  -  Proof  of  principle:  Tier  one  tests  should  document  the  efficacy  of  the 
incorporated biocide in the relevant matrix against relevant target organism(s) under 
relevant conditions (e.g. humidity, temperature). 

•  Tier  2  -  Simulated  Use:  Tier  two  tests  should  document  the  efficacy  of  the 
incorporated  biocide  in  the  relevant  matrix  under  real-life  conditions  (e.g.  way  of 
contamination, cleaning regimes, time to take effect) and the duration of the effect. 

Depending  on  the  claim  made  (e.g.  “kills  bacteria  on  door-handles  to  prevent  cross 
contamination”, “protects against mosquito-bites”), even Tier 3 testing can be necessary: 

•  Tier  3  -  In-Use  Evaluation/Field  studies:  To  substantiate  health  benefit  claims, 
treated and untreated articles would be tested via statistically designed use trials by 
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Generally,  the  principle  applies  that  only  claims  can  be  made  which  have  been 
demonstrated. 

5.4.6.1 Determining the purpose of the Treatment 

The  effects  of  articles  with  a  disinfection  claim  cannot  be  detected  by  changes  in 
appearance, mechanical properties or odour. The precondition for demonstrating efficacy is a 
clear  description  of  the  purpose  of  the  treatment.  Often,  claims  are  unclear  about  whether 
the treatment prevents growth or kills bacteria on contact. On most articles, no bacteria will 
grow  under  normal  conditions  of  use.  Nevertheless,  antibacterial  claims  (such  as  ‘anti-
bacterial’, ‘hygienically clean’, ‘free of bacteria’, ‘prevents the spread of hazardous bacteria’) 
are  made,  insinuating  that  bacteria  will  be  killed  on  the  material,  though  only  growth 
inhibition tests have been carried out. In most environments, the sheer presence of bacteria 
does not present a problem. If this is a problem, it is in most cases much more effective to 
use traditional disinfection methods with a liquid disinfectant. In most cases, the treatment 
of articles  should not be used as the  only measure of disinfection, but should be combined 
with a disinfection management regime. 

5.4.6.2 Effects Intended to Inhibit Microbial Growth 

Under  the  majority  of  indoor  situations,  most  micro-organisms  will  not  grow  on 
environmental surfaces due to lack of humidity. To make a claim for growth inhibition, wet 
or  at  least  humid  conditions  are  a  precondition,  unless  otherwise  justified.  To  demonstrate 
such a claim, sub-samples of treated and untreated material of the article in question could 
be  tested  using  a  method  adapted  from  ISO  22196  (see  Figure  3).  Soiling  conditions, 
temperature,  test  species  and  contact  time  have  to  be  adapted  to  mimic  a  realistic  in-use 
situation  (Tier  1).  The  impact  of  in-use  conditions  like  ageing  or  cleaning  regimes  on  the 
effect  would  have  to  be  included  in  the  testing  (Tier  2).  The  minimum  requirements  for 
disinfection  are  laid  down  in  the  Claims  matrix  for  treated  articles  (see  Appendix  1)  with 
claims to protect humans or  animals [http://echa.europa.eu/about-us/who-we-are/biocidal-
products-committee/working-groups/efficacy]. 

 
 
 
 
 
   
 
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Figure 3: A Test for Antibacterial Activity in Wet Conditions 

ISO 22196 

Inoculate 3 Test Pieces (50 x 50 mm) 
with 400 µl of Cell Suspension Each 

Cover with Sterile 
Polyethylene  Film  
(40 x 40 mm) 

Prepare 
Cell suspension 
-1

6
 cells ml

(ca 10

) 

Transfer each 
Film and Test Piece 
to Neutraliser in 
Stomacher Bag 

Incubate for 24 Hours at 35°C 
Under Humid Conditions 

Polyethylene Film 

Cell Suspension 

Test Piece 

Determine CFU 

ISO 22196, Method Outline: 

An aliquot (usually 400 μl) of a log phase bacterial cell suspension (ca105 cells ml-1) in 1/500 
Nutrient  Broth  are  held  in  intimate  contact  with  each  of  3  replicates  of  both  treated  and 
untreated variants of the test materials using a 40 mm x 40 mm polyethylene film (e.g. cut 
from  a  sterile  Stomacher  bag)  for  24  hours  at  35°C.  Usually,  S.  aureus,  P.  aeruginosa, 
Enterococcus hirae and E. coli should be tested (see Appendix 3). The populations are then 
recovered  using  a  neutraliser  solution  and  the  size  of  the  surviving  populations  are 
determined  as  colony  forming  units  (CFUs)  using  a  dilution  plate  count  method.  Additional 
replicate  unfortified  samples  are  also  inoculated  in  the  same  manner  but  are  analysed 
immediately  to  determine  the  size  of  microbial  population  present  prior  to  incubation.  The 
differences  between  the  initial  and  final  population  as  well  as  between  the  treated  and 
untreated  materials  are  used  to  assess  the  basic  antibacterial  properties  of  the  test 
materials. 

5.4.6.3 Effects intended to Kill Micro-organisms through Contact 

Claims made for materials and articles to kill on contact to prevent cross-contamination are 
not easy to demonstrate.  Mostly, the effect will require the  release  of  the active substance 
from  the  surface  of  the  material;  this  release  needs  to  be  triggered  somehow.  In  the 
majority of cases, water or other liquids are the crucial component to facilitate such release 
and  transfer.  If  the  event  that  caused  the  deposition  of  the  target  organism  does  not 
introduce moisture and the normal exposure conditions of the material or article are dry (or 
only  subject to normal, ambient indoor humidity), the  effect  of the treatment will probably 
be limited. 

Another issue is the speed of activity needed to inhibit cross-contamination. If for instance 
door  handles  in  a  hospital  would  be  treated  with  an  active  substance  to  kill  deposited 

 
   
 
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pathogenic  organisms,  the  effect  would  have  to  be  sufficiently  fast  to  prevent  the  next 
person  using  the  door  handle  from  cross-contamination.  In  combination  with  the  little 
moisture  which  is  deposited  in  the  event,  it  will  be  challenging  to  demonstrate  a  satisfying 
effect.  The  minimum  requirements  for  disinfection  are  laid  down  in  the  Claims  matrix  for 
treated  articles  (see  Appendix  1)  with  a  claim  to  protect  humans  or  animals.  Additional 
requirements may apply depending on the claim made. 

Testing could be carried out using protocols such as those given in Figures 4, 5 and 6 below. 
Again,  care  must  be  taken  to  adapt  test  conditions  to  realistic  in-use  conditions.  Figures  4 
and  5  show  the  approach  used  for  non-porous  materials  and  for  absorbent  materials, 
respectively,  both  intended  to  simulate  contamination  through  contact  with  splashes  of 
contaminated  liquids.  Figure  6  illustrates  a  protocol  intended  to  simulate  contamination 
through, for example, hand/gloved hand contact. 

5.4.6.4 Acceptance Criteria 

The  performance  criteria  for  treated  articles  can  be  found  in  the  Claims  Matrix  for  treated 
articles  (Appendix  1).  For  choosing  test  organisms  please  refer  to  the  liquid  disinfectants 
(Appendix  3).  As  the  performance  criteria  for  treated  articles  are  lower  than  for  liquid 
disinfectants, the treatment of articles should generally not be used as the only measure of 
disinfection, but should be combined with a disinfection management regime. 

 
 
 
 
 
   
 
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Figure 4: Simulated Splash Model Non-Porous Materials 

CFU= colony forming units 

Prepare  replicate  test-pieces  (4  per 
contact time per treatment) 

Inoculate Test Pieces (30 x 30 mm) 
with 100 µl of Cell Suspension each 

Incubate  without cover at 24°C for 
5 -60 min at 60% Relative Humidity 

Cell suspension 
-1
) 
(ca 10

6
 cells ml

Constant Humidity Chamber 

Transfer Test Piece 
to Neutraliser in 
Stomacher Bag 

Determine CFU for 
each contact time 

 
   
 
 
 
 
 
 
 
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Figure 5: Simulated Splash Model Porous Materials 

CFU= colony forming units, RH= relative humidity,  

BSA= Bovine Serum Albumine 

Prepare replicate swatches (4 
per contact time per 
treatment) and place on 
sterile grid to allow good air 
circulation 

Suspension (ca 10

5
 CFU ml

-1

species in BSA solution (15 g litre

) of test 
-1
) 

Swatches inoculated 
with 100 µl 

Determine CFU 

Incubated for 5 – 60 
min at 24°C & 50% 
RH 

Individual swatches 
transferred to 10 ml 
neutraliser at intervals 

 
 
 
 
 
   
 
 
 
 
 
Figure 6: Printing Model 

TVC= total viable count 
CFU= colony forming units 

Prepare 
Cell suspension 
-1

5
 cells ml

(ca 10

) 

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Inoculate Large Bioassay Dish 
containing 
/10 strength Nutrient 
Agar with10 ml of cell suspension 

1

Incubate for 
18 Hours at 25°C 

Transfer to 
Test Pieces in Pairs 
1000 g 

Lift Cells from 
Bioassay Plate 
100 g 

Recover cells in 
Neutraliser and 
Determine TVC 

Determine TVC Prior  
to Incubation 

Incubate Inoculated Test Piece at 
20°C for 5 min then Determine CFU 

Transfer Inoculum to Test Pieces 

Locking ring 

Plate replicator 

 
   
 
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Table 9: Protection of Humans or Animals – Example Claims, Problems and Testing 
Approaches 

Claim 

PT  Proof required 

Example method 

Bedside cabinet for 
use in hospitals that 
has been treated to 
reduce infections by 
killing ‘bacteria on 
contact’. 

2  Data should show that micro-

organisms, when deposited through 
skin contact (even under simulated 
conditions) and through the 
deposition of fine aerosols are killed 
within a time-frame that would 
prevent the surfaces becoming a 
vector for cross-contamination. 

Plaques made of the identical 
material used for the cabinet 
are employed in the test. Both 
treated and untreated variants 
are used.  

Skin contact 

Aerosol 

A plastic conveyer 
belt is treated to 
prevent the growth 
of bacteria between 
cleaning intervals in 
a food factory. 

4  Data should show that relevant 
bacteria grow on an untreated 
conveyer belt under normal conditions 
of use during a 6 hour interval. 
Significantly reduced growth should 
be demonstrated on the treated belt. 

The method described in 
Figure 6 is employed to 
deposit bacteria onto test 
plaques. A range of contact 
times between 5 minutes and 
1 hour are used. A log 
reduction of 3 should be 
achieved. 

The method described in 
Figure 4 is adapted for use by 
employing multiple droplets of 
1 µl on each test plaque. A 
range of contact times 
between 5 minutes and 1 hour 
are used to explore activity. A 
log reduction of 3 should be 
achieved. 

Plaques made of the identical 
material used for the belt are 
employed in the test. Both 
treated and untreated variants 
are used. 

ISO 22196 is adapted to 
simulate a moist conveyor 
belt. A soiling agent relevant 
to the end use is included. A 
contact time and temperature 
equal to that encountered in 
practice are employed. 

 
 
 
 
 
   
 
 
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Table 10: Basic Requirements for a Valid Test Protection of Humans or Animals 

The following summary provides a guide to the basic requirements for a valid test: 

i. 

ii. 

The test should be carried out on the type of final article. 

A test which mimics the way of deposition and the type of material needs to be 

chosen. 

iii. 

An untreated variant of the test material must be included such that the impact 

of the treatment can be demonstrated. 

iv. 

Test conditions should reflect normal conditions of use in terms of humidity, 

temperature, soiling, contact frequency, etc. 

v. 

The test should employ organisms that are relevant to the end use of the article 

and the purpose being claimed. 

vi. 

Tests that employ a single species of organisms should be favoured over those 

that use consortia. 

vii.  Minimum of three replicate test pieces of both treated and untreated materials 

should be employed (unless justified). 

viii. 

The final data should include either some indication of the impact of service 

conditions on the performance of the treated material/article or data from an 

ageing study. The intention is to demonstrate how long the claimed effect will be 

sustained. 

ix. 

If claims are made which require a field test, relevant data including statistical 
evaluations have to be provided. 

 
   
 
 
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140 

5.5 Preservatives (Main group 2) 

General 

Preservatives in main group 2 are intended to prevent the biodeterioration of a material or a 
matrix.  Wood  can  lose  stability  by  the  action  of  micro-organisms  or  insects,  fabric  can  be 
destroyed  by  fungi,  and  even  polymer-based  plastics  are  prone  to  biological  deterioration. 
Plasticised  PVC  would  soon  become  fouled  by  surface  growths  of  fungi,  lose  plasticity  and 
crack  without  the  inclusion  of  a  fungicide.  A  water-based  paint,  free  of  volatile  organic 
compounds  (VOCs),  could  not  be  stored  without  the  use  of  a  biocide.  Polyurethane,  for 
example as used for the soles of shoes, can become colonised by fungi and actinomycetes. 
The  heat  exchangers  in  cooling  towers  have  to  be  kept  free  from  microbial  growth  to 
enhance performance by treatment of the cooling liquid. 

This  section  covers  the  group  of  preservatives  (PT6  to  PT13)  and  the  following  sections 
(5.5.1-5.5.3)  apply  to  all  PTs  (or  as  indicated  in  the  headings).  For  PT8,  the  guidance  is 
more developed and includes standard tests, which is not the case for the other PTs: PT8 is 
the exception and section 5.5.8 is dedicated to PT8. 

5.5.1 Distinction between preservation/curative treatment and disinfection 

Preservatives  are  directed  towards  the  protection  of  a  material.  If  the  material  itself  is  not 
affected  by  the  target  organisms,  the  claim  does  not  belong  in  main  group  2.  The  aim  of 
preservation is to prevent microbial spoilage, decay  or the accumulation  of biomass that is 
detrimental  to  the  functionality  of  an  item,  material  or  system.  Detrimental  effects  can  be 
caused by proliferation of cells or by the metabolic activity of cells and may not necessarily 
involve  cell  multiplication.  The  presence  of  micro-organisms  can  result  in  either  a 
degradation of the matrix in which they are present or damage to the system in which they 
are present either due to their metabolic activities (e.g. corrosion) or by fouling or blocking 
pipes,  forming  biofilms  on  heat  exchangers  etc.  It  is  not  the  intention  of  preservatives  to 
transfer  their  effects  to  other  materials,  humans  or  animals,  but  to  protect  the  material 
itself. A long-term effect is generally required. A preservative can have a reversible effect on 
micro-organisms  (e.g.  by  causing  stress  or  cell  damage  without  total  loss  of  viability).  In 
contrast to disinfection no level of reduction is defined for a set of predefined claims. 

Curative treatments are also directed towards material protection and therefore likewise fall 
into main group 217. The aim of a curative action is to either cure microbial spoilage which 
has already occurred or to eliminate / reduce populations in materials and systems prior to 
them being treated with a preservative (in some instances a biocidal product can have both 
curative and preservative functionality). 

The  level  required  to  prevent  spoilage  in  different  media/conditions  will  be  defined  by  the 
individual claim made. This will also be the case when the treatment is intended to achieve a 
curative action. 

The claim made will define in which of the PTs an application will fall. The following data is 
needed: 

•  A  problem  description:  Scale,  speed  and  type  of  effect  required  and  what  would 

happen if the biocide was not present 

•  The target organisms 

17 See CA-Sept15-Doc.8.3 – Curative use of preservatives 

 
 
 
 
 
   
 
 
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•  Categorisation of the material/matrix to be treated including dose-rate/concentration 

of the biocide in the material/matrix. 

•  The  intended  use  pattern  of  the  treated  material/matrix  including  service-life, 

weathering conditions, leaching (intended or unintended). 

Figure 7: Decision scheme for the distinction between preservation/curative action 
and disinfection 

Is  the  treatment 
intended  to 
protect  the  material/article  or  its 
functionality 
biological 
deterioration  during  storage  or  in 
service,  extend  its  durability  or 
prevent odour? 

from 

Is  the  active  substance/product 
intended for curative treatment of 
wood, industrial liquids, solutions, 
dispersions or processes? 

Is  the  treatment  intended  to 
protect  humans  or  animals?  Is 
remedial 
of 
treatment 
construction  materials 
with 
algaecides intended? 

Preservative (Main Group 2) 

Disinfectant (Main Group 1) 

5.5.1.1 Curative uses 

Curative uses often require rates and speeds of effects that are similar to those required for 
disinfectants but do not have prescribed performance standards (with the exception of some 
PT8  standards).  Such  uses  are  nevertheless  intended  to  cure  (eliminate  or  reduce) 
contamination  in  materials,  matrices  or  systems.  They  therefore  fall  under  main  group  2. 
Performance  requirements  will  be  defined  by  the  requirements  of  either  the  matrix  or  the 
process  involved.  A  curative  effect  and  a  preservative  effect  may  sometimes  be  achieved 
using  the  same  biocidal  product,  only  the  concentration  may  differ.  In  other  cases  active 
substances  with  curative  properties  will  be  combined  with  those  that  have  preservative. 
Curative and preservative effects need to be demonstrated separately and different methods 
need to be employed. When claims are made for curative uses, it is important to carry out 
the health and environmental risk assessment with any higher doses that may be required. 
A  typical  example  of  curative  action  is  the  treatment  of  a  contaminated  product  prior  to 
packaging and sale (in some cases in addition to a preservative – in other cases the curative 
product  may  be  capable  of  achieving  both  a  preservative  and  a  curative  effect).  Another 
example is the treatment of a contaminated system by reducing the microbial population it 
contains to limits that are acceptable to the process (e.g. on a paper mill). Please read more 
about testing of curative uses in section 5.5.5.1 and 5.5.8. 

 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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5.5.1.2 Borderline case: Algaecides 

If algae are expected not to destroy the material or damage its function, algaecides are not 
considered to be preservatives. Thus products used against algae for treatment of swimming 
pools, aquariums and other waters and for the remedial treatment of construction materials 
belong  to  product  type  2  in  main  group  1,  whereas  products  with  protective  function  are 
considered as products belonging to main group 2. 

For example surface coatings for outdoor use are often formulated with both a fungicide and 
an algaecide. The algaecide, like the fungicide, is performing a preservative  function in the 
coating and is thus covered by PT 7. Similarly, algaecides are incorporated into plastics (e.g. 
electricity  pylon  insulation  sleeves  -  to  prevent  growth  that  would  otherwise  cause  arcing 
and system failure) and material used in aquatic and marine environments (including some 
cementitious  materials).  Algae  are  a  problem  in  many  water-based  cooling  systems  and 
water-based process systems (e.g. paper making), where either a preservative or a curative 
action may be required. Such applications likewise belong to main group 2. 

5.5.1.3 Borderline cases: Treated articles 

Treated articles can both belong to Main Group 2 or Main Group 1 (and even to Main Group 
3  or  4).  Please  refer  to  section  5.  3  on  treated  articles  and  section  5.4.6  on  materials  and 
articles treated to protect humans or animals. 

5.5.2 Principles for testing preservatives 

The  aim  of  any  preservation  is  to  maintain  the  present  state/properties  of  a  material  or 
matrix  along  with  it  its  functionality.  This  can  be  done  in  several  ways:  To  determine 
microbial activity in a biocide-free material, the method of measuring colony forming units is 
the most common approach to prove that a preservative is needed, i.e. the population needs 
to be shown to increase in size in the untreated material. The production of a biofilm or an 
increase  in  biomass  may  also  be  appropriate.  Other  parameters  indicating  metabolism  can 
also  be  documented  like  e.g.  changes  in  pH,  in  viscosity,  in  colour.  Data  needs  to  be 
recorded from the beginning of the test (incubation time 0) and before  and after each new 
inoculation. 

Showing  growth  /  metabolism  of  the  micro-organisms  in  the  untreated  system  is  an 
essential  requirement  of  any  demonstration  of  effectiveness  of  an  active  substance  or 
biocidal  product.  It  is  then  assumed,  if  not  proven  in  every  case,  that  changes  have  taken 
place that were induced by microbial growth and that this can be prevented by the use of a 
biocide acting as a preservative. Often, when growth cannot be proven this is caused by an 
unnecessarily  high  inoculation  rate.  If,  at  the  beginning  of  the  test,  an  inoculum  of  for 
example  104  CFU  for  bacteria  is  employed,  an  increase  to  105  -  106  can  often  easily  be 
shown during the test period. When a higher inoculum density for example 106 is employed, 
growth  is  much  harder  to  achieve  due  to  limitations  in  the  supply  of  nutrient  etc.  An 
important  consideration is to use  a model  substrate that can support  growth readily rather 
than  attempt  to  achieve  growth  in  a  final  product  that  is  less  susceptible  to  the  non-
acclimated species employed in laboratory tests (i.e. it is often nearly impossible to replicate 
the failure phenomena observed in practice in a laboratory). 

Often a fungicidal or bactericidal claim needs to be supported. For this purpose a species can 
be  tested  singly  or,  as  it  is  good  practice  in  many  test  protocols,  in  mixed  suspensions  of 
either  bacterial  species  or  fungal  species.  Mixing  of  bacteria  and  fungi  should  generally  be 
avoided  in  these  suspensions,  but  filamentous  fungi  (“moulds”)  and  non-filamentous  fungi 

 
 
 
 
 
   
 
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(“yeasts”) can be mixed in the inoculum. However, for determining growth different methods 
need to be applied for yeasts and filamentous fungi. 

Many  micro-organisms  are  able  to  form  dormant  cells  or  spores  to  survive  unfavourable 
environmental  conditions.  These  resting  cells  do  not  proliferate  and  show  no  significant 
metabolic  activity  until  they  find  a  suitable  environment.  It  is  therefore  possible  that 
vegetative and active cells, being exposed to an unfavourable environment e.g. a synthetic 
paint containing solvent or a preservative, are forced into dormancy. Only when a sample of 
the material is taken out of this environment  and is spread  onto a nutrient medium do the 
cells start to grow and to build new colonies. This underlines that the appearance of colony 
forming  units  (CFU)  on  a  nutrient  media  is  not  necessarily  sufficient  evidence  that  growth 
had  been  occurring  in  the  matrix  used  in  the  test.  Growth  can  only  be  determined  by 
counting CFU and demonstrating that the number of CFU increased in the untreated matrix 
during  incubation,  compared  to  the  number  measured  immediately  after  inoculation.  The 
same  or  a  smaller  number  of  CFU  than  measured  initially  demonstrates  survival,  but  not 
necessarily growth. However, for testing solid material, showing growth by adding a nutrient 
medium  to  the  material  is  not  necessarily  enough.  It  needs  to  be  shown  that  the  material 
itself  is  damaged  or  loses  its  functionality,  or,  alternatively,  provides  growth  of  micro-
organisms relevant for the group of organisms which have a negative impact on the stability 
and/or functionality of the material. Please read more in section 5.5.7. 

5.5.3 Tiered approach to testing preservatives 

A tiered approach should be followed for testing biocidal products: 

Tier 1 -  Proof  of  principle:  Tier  one  tests  should  document  the  biocidal  efficacy  of  the 
incorporated  biocide  in  a  relevant  model  matrix  against  the  target  organism(s) 
under relevant basic environmental conditions (e.g. temperature, humidity). 

Tier 2 -  Simulated  Use:  The  biocide  should  demonstrate  efficacy  under  real  life  conditions 
relevant  to  its  anticipated  service  life.  Factors  such  as  weathering,  UV-stability, 
extended ageing or leaching should be considered. 

Tier 3 – In-use  evaluation/field  studies:  to  substantiate  specific  claims,  treated  and  control 
articles/products  can  be  tested  via  statistically  designed  in-use  trials  by  a 
representative user group, or by other appropriate methods. 

In a Tier 1 test, the damage should be shown in a model matrix and demonstrate how the 
inclusion  of  the  biocide  prevents  it  (often  with  the  help  of  an  inoculum  representing  the 
organisms  that  cause  the  damage).  In  a  Tier  2  test,  damage  or  impact  of  the  target 
organisms  under  either  simulated  use  conditions  or  in  a  manner  that  simulates  an 
anticipated shelf life should be shown, and even sometimes without the use of an inoculum 
(soil burial). When moving up from tier 1 to tier 2, a test design has to be more tailored to 
the  field  of  application  envisaged.  In  tier  1,  existing  standards  are  often  suitable  when  the 
biocide  is  tested  in  a  relevant  matrix  with  defined  organisms  and  under  relevant  and 
reproducible  conditions  (which  are  normally  only  to  be  found  in  a  laboratory).  In  tier  2, 
testing is more complex and often specific standards do not exist. However, sometimes the 
same standards can be used as for tier 1 tests, simulating use conditions by employing pre-
treatment  of  the  matrix.  There  may  be  a  need  for  weathering  cycles,  wind  tunnel  tests, 
cleaning regimes etc. Similarly soiling and the influence of other micro-organisms can be of 
more significance. Accelerated aging tests may have to be performed before microbiological 
testing  to  allow  for  factors  such  as  UV,  temperature  changes,  leaching  etc.  Consideration 
must be given to which environmental conditions are relevant for simulated aging in realistic 
in-use  conditions.  When  aging  is  performed  in  the  field  or  under  in-use  conditions, 

 
   
 
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reproducibility  can  become  a  difficult  issue,  as  the  aging  factors  such  as  e.g.  evaporation 
and soiling are difficult to reproduce and can influence the results.  Generally, the applicant 
should  be  able  to  justify  how  the  specific  conditions  used  in  testing  relate  to  the  in-use 
conditions relevant to the product or active substance. Tier 3 testing entirely depends on the 
claim made and is generally for specific uses in case of specific claims. The results have to 
be relevant for that claim and to be scientifically sound. 

5.5.4 Standard Test Methods 

A  list  of  the  most  commonly  used  standard  test  methods  can  be  found  in  Appendices  8,  9 
and  10;  however,  please  note  that  these  test  methods  are  not  necessarily  appropriate  to 
use; they are listed  with comments to give an orientation for the assessor as to  when and 
where  these  tests  can  be  meaningful  to  prove  /support  a  claim  and  when  they  aren’t.  In 
contrast  to  disinfection,  there  are  no  specific  tests  allocated  to  the  different  tiers,  with  the 
exception  for  PT8  where  standard-tests  are  available  and  tiered  testing  is  defined,  (see 
section 5.5.8 for more information). Often the same test can be employed for tier 1 and tier 
2,  and  only  the  pre-treatment  of  the  matrix  will  differ.  Different  factors  can  trigger  the 
choice of a test: In some cases the choice of one type of method over another is related to 
the  speed  with  which  it  generates  results.  Often,  a  method  is  ‘known’  to  be  capable  of 
guiding the choice and  concentration  of  a biocide for a certain material through experience 
within an industry. However, this may not necessarily mean that the method is suitable for 
demonstrating the claim made. 

Care  has  to  be  taken  as  to  whether  the  test  method  is  appropriate  for  the  testing  of 
preservatives,  or  if  it  is  intended  to  prove  a  curative/sanitising  activity  of  a  biocide. 
Generally, for preservative action growth needs to be shown in the untreated controls. The 
number  of  replicates  required  by  the  methodology  is  not  necessarily  3  replicates;  in  such 
cases this needs to be explained and justified. 

Nevertheless,  an  existing  test  method  can  form  a  good  basis  regarding  the  parameters  of 
choice  of  micro-organisms,  temperature,  and  choice  of  neutraliser.  If  necessary,  these 
methods  need  to  be  amended  by  adding  untreated  control  samples,  determining  the 
numbers  of  organisms  that  can  be  recovered  immediately  after  inoculation  (0  hours 
incubation), use of a neutraliser, and the use of a smaller sized inoculum etc. Particularly for 
tier  2  and  3  testing,  it  is  important  that  the  chosen  adaptations  reflect  the  relevant 
conditions for which the claims must apply. 

Specific tests which are recommended for certain uses are described under the  sections for 
the different PTs. 

5.5.4.1 Practical aspects for testing bacteria 

A relevant study that proves the need for a biocide and its efficacy as a preservative against 
bacteria must have the following features: 

a.  The test must be performed in a range of relevant model matrixes that the claim of 

efficacy is made for (e.g. dishwasher liquid, paints, glues, textiles, etc); 

b.  The  test  has  to  be  performed  in  relevant  environmental  conditions  (temperature, 

type of matrix, humidity); 

c.  Control samples without the addition of a biocide must be included during the whole 
test. These control samples must be handled identically to the other samples, except 
that  they  must  have  no  biocide  included.  The  study  must  include  replicate  sub-

 
 
 
 
 
   
 
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samples for each treatment (minimum of 3; if less than 3 replicates, then explain and 
justify). 

d.  For  preservative  uses,  the  control  samples  should  typically  show  growth  (e.g. 
indicated  by  an  increased  number  of  CFUs)  during  incubation  and  this  has  to  be 
documented. If no growth in the control samples can be seen, this could indicate that 
only the dormant stages of bacterial cells, without active metabolism, are present in 
the  matrix.  The  treated  samples  should  show  statistically  significant  effects  as 
compared to the controls; 

e.  Only  if  growth  cannot  be  proven  by  increase  in  CFU,  data  concerning  other  factors 
like e.g. CO2-emission, O2 depletion, change of pH, colour change or disintegration of 
the matrix should be used to demonstrate the need of preservation of a matrix by the 
active ingredient or preservative; 

f.  Relevant bacteria for the intended use have to be tested. 

5.5.4.2 Practical aspects for testing fungi 

A relevant study that proves the need of a biocide and its efficacy as a preservative against 
filamentous fungi is in many ways the same as for bacteria, but an attempt to count colony 
forming  units  of  thread-like  mycelia  after  incubation  in  liquid  systems  is  bound  to  fail  for 
several reasons:  

• 

It is impossible to take a  representative aliquot from the incubated test vessel since 
the mycelia tend to conglomerate into pellets of different sizes (often blocking the tip 
of a pipette). 

•  Different  seized  fragments  of  mycelium  and  spores  that  are  dormant  in  the  matrix 
form  colonies  on  a  petri  dish  and  their  origin  cannot  be  differentiated  and  so  their 
numbers do not reflect the increase in biomass that has occurred. 

However,  counting  CFU  is  a  practical  option  to  measure  the  recovery  rate  of  spores 
inoculated into liquids before spore germination (time 0 analysis) and for unicellular yeasts. 
At this stage, no mycelia have formed in the liquid, so no fragments will be counted as CFU 
and  wrongly  interpreted  as  growth.  Therefore,  after  the  control  samples  and  the  biocide-
containing samples have been inoculated with spores, the recovery rate can be recorded by 
measuring colony forming units. 

Ascomycetes  and  fungi  imperfecti  form  thread-like  hyphe  and  spores.  Spores  serve  as 
dormant  stages  when  environmental  conditions  are  detrimental  to  growth.  When  growth 
conditions  are  favourable,  the  spores  germinate  and  form  a  mycelium  and  maybe  other 
spores. In liquids the fungal growth tends to form pellets. These can be very small or up to 
several  millimetres  in  diameter.  Furthermore,  it  is  possible  that  a  visible  biofilm  will 
accumulate at the sides of the test vessel, e.g. an Erlenmeyer flask or on the surface of the 
matrix.  Both  phenomena  are  visible  by  the  naked  eye  and  clearly  demonstrate  that  the 
fungus  has  grown.  In  highly  fluid  materials  this  growth  can  be  quantified  by  filtering  the 
whole contents of the test vessel and then determining the amount of growth as dry weight. 
The use of replicates is an important factor in such tests. The number of replicates required 
by  the  methodology  is  not  necessarily  3  which  is  the  usual  minimum;  in  such  cases  this 
needs to be explained and justified. 

For  testing  solid  materials,  fungal  growth  is  often  assessed  by  optical  appearance,  using  a 
rating scale from 0 (no growth) to 5 (>70% cover). 

 
   
 
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5.5.5 Testing conditions for specific states  

5.5.5.1 Wet-state preservation and curative treatments  

Preservation (PT 6, 13) 
Challenge  tests  are  generally  employed  for  preservatives  which  must  preserve  liquid 
matrices, dispersions or fluids used in systems. The inoculum used and the strength of the 
inoculum depends on which claim must be supported. For preservation claims, growth needs 
to  be  shown  in  the  untreated  samples  and  prevention  of  growth  in  the  treated  samples.  A 
larger  population  (generated  by  prior  growth  in  an  untreated  matrix)  may  be  more 
appropriate  for  demonstrating  a  curative  effect.  Some  methods  for  wet-state  preservation 
are  compiled  in  Appendix  8,  however,  please  note  that  these  test  methods  are  not 
necessarily appropriate to use; they are listed with comments to give an orientation for the 
assessor as to when and where these tests can be meaningful to prove /support a claim and 
when they aren’t. 

A  series  of  concentrations  of  the  active  substance  or  the  biocidal  product  should  be 
employed  in  order  to  investigate  which  concentration  achieves  which  level  of  efficacy.  It  is 
likely that the  application  rate in practice will  vary depending on the  in-use conditions  of a 
biocidal product even though the matrix is identical, e.g. in a metal working fluid, where the 
in-use concentration is achieved by diluting the product at the point of use. 

Curative Treatments (PT 6, 7, 11, 12, 13) 
Suspension  tests  are  generally  employed  for  curative  treatments  of  liquid  matrices, 
dispersions  or  systems.  A  curative  treatment  might  be  applied  to  a  system  to  reduce  a 
population prior to employing a maintenance regime / treatment (e.g. PTs 11, 12 and 13) or 
it might be used prior to the addition of a preservative in either a final product, intermediate 
or a raw material (e.g. PT 6). A model matrix that has been inoculated with micro-organisms 
appropriate to the claim to achieve either growth or a stable population must be treated with 
the active substance / biocidal product and the effect measured after an appropriate contact 
time  using  a  dilution  plate  count  (methods  described  for  wet  state  preservation  can  be 
employed  to  generate  the  model  contaminated  matrices  /  systems).  The  inoculum  can 
comprise of aerobic or anaerobic bacteria, endospore forming bacteria, yeasts, fungal spores 
and / or mycelial growth as appropriate to the claim. A log-reduction relevant to the matrix 
and its use needs to be shown in the treated samples. Viability / growth should be shown to 
be  maintained  in  the  untreated  samples.  Replicate  sub-samples  must  be  employed 
(minimum  of  3,  but  if  the  number  of  replicates  required  by  the  methodology  is  not  3  this 
needs to be  explained  and justified) and any  differences that result should be  shown to be 
statistically  significant.  Data  from  samples  treated  under  field  conditions  can  be  used  as 
supporting  evidence  provided  that  any  effects  shown  can  be  attributed  to  the  treatment 
applied. 

5.5.5.2 Protection of solid material: PT 7, 9, 10 

This  section  describes  the  nature  and  extent  of  data  which  should  be  made  available  to 
support  the  label  claims  for  biocidal  products  within  PT  7  through  PT  10.  The  common 
denominator  of  these  PTs  is  that  they  concern  the  treatment  of  solid  material  where  use 
conditions can vary considerably, depending on the site and type of use of the material (e.g. 
treated wood to be used in constant contact with water compared to use in dry conditions; a 
film preservative to protect a bathroom sealant compared to protecting a house-façade). In 
contrast  to  liquid  disinfectants  or  preservatives  belonging  to  PTs  11,  12  and  13,  where 
application  often  takes  place  on-site  (that  is  where  the  target  organisms  occur),  the 
treatment  of  materials  can  take  place  anywhere,  for  example  where  the  material  is 
manufactured or at a specific-treatment site. This may not necessarily be within the EU. 

 
 
 
 
 
   
 
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Use  conditions  are  much  more  variable  for  these  product  types  than  they  are  for  liquid 
disinfectants  and  liquid  preservatives.  Often,  many  different  materials  can  be  treated  with 
the  same  biocide,  and  even  more  different  articles  can  be  manufactured  from  the  treated 
materials,  which  are  used  in  a  wide  variety  of  conditions.  For  instance,  water  absorption 
properties  of  different  polymer  materials  vary  and  so  does  the  release  of  the  biocide.  The 
concentration  of  the  biocide  has  to  be  adapted  accordingly.  Biocides  can  be  applied  as  a 
coating  to  fabrics  or  can  be  incorporated  into  the  material  by  adding  the  biocide  to  the 
polymer before spinning or  extrusion.  This alters the fixation in or on the material and has 
an  impact  on  performance.  Materials  and  articles  can  be  used  indoors,  outdoors,  in  wet, 
humid  or  dry  conditions  and  at  varying  temperatures.  All  of  this  has  an  impact  on 
performance.  Simulating  service  life,  as  length,  weathering  conditions,  temperature, 
leaching,  laundering,  etc.  is  crucial  for  testing  of  products  within  these  PTs.  Thus,  efficacy 
testing  for  PT  7  through  10  requires  a  good  description  of  the  frame  in  which  the  biocide 
must  perform.  In  many  cases  it  will  be  impossible  to  test  every  material/substance 
combination;  it  might  be  feasible,  however,  to  categorize  different  parameters:  material, 
concentrations  ranges,  use  (outdoor,  indoor,  temperature,  humidity,  use  for  load-bearing 
components,  etc.)  and  to  try  to  test  representative,  preferably  worst-case,  examples  for 
every  category.  It  is  important  though,  to  describe  and  justify  which  range  the  tested 
sample represents. 

Model matrices 
The array of possible material and biocide combinations is vast and phenomena observed in 
practice  cannot  always  be  reproduced  in  the  laboratory.  A  model  matrix  has  to  be  chosen 
which  represents  a  certain  type  of  material  and  which  is  relevant  to  the  intended  use.  For 
example,  plasticised  PVC  and  polyurethane  would  be  useful  models  for  rigid  or  semi-rigid 
polymers  and  a  room  temperature  vulcanised  silicone  would  provide  a  useful  model  of  a 
sealant etc. Relevance is the key factor.  Thus,  if a treatment is intended to protect natural 
fibres in service then a natural fibre should be employed as the model. When more than one 
type of material (e.g. plastics, paints and synthetic fibres) can be protected by the biocide, 
then representative matrices that demonstrate the range of protection should be employed. 
Different  materials  can  require  different  biocide  concentrations  due  to  varying  release 
behaviour.  It  is  also  important  to  consider  what  the  purpose  of  the  end  use  is  (e.g.  in  one 
application  the  biocide  may  provide  essential  protection  of  a  matrix  whereas  in  another  it 
may increase durability). The objective is in any case to support the claims made. 

Representative species 
The  species  employed in any test should be relevant to the intended  use (i.e. fungi  should 
be  employed  if  the  material  is  affected  by  fungal  growth,  odour  producing  bacteria  to  be 
found  on  the  skin  should  be  employed  for  odour  testing,  etc.).  Consortia  rather  than 
individual  species  should  be  employed  (although  mixing  bacteria  with  fungi,  algae  etc. 
should, in general, be avoided, see 5.5.2). In exceptional cases, it can be acceptable to use 
individual  species  when  justified,  however,  using  consortia  of  micro-organisms  can  be  a 
good  option  to  reflect  realistic  use  conditions  but  the  use  of  individual  species  is  also 
acceptable.  The  species  employed  in  the  tests  should  be  relevant  to  the  material  under 
investigation especially where the prevention of the degradation of a material is intended. In 
many  cases  the  organisms  will  be  specified  with  the  method.  Very  limited  ranges  of  model 
organisms  should  be  avoided  where  possible  (e.g.  the  use  of  A.  brasiliensis  as  the  sole 
fungus).  The  test  should  include  replicates  (at  least  three)  for  both  the  treated  and 
untreated variants. 

 
   
 
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Table 11: Examples 

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Claim 

PT 

Example Problem 

Example Method 

Fungicide is used to treat 
paint to prevent causing 
stains by mould growth 
in service 

7 

Painted panels exposed to 
weather become stained by 
mould growth and have to be 
re-painted more often. 

BS 3900 Part G6 

Painted panels inoculated with 
a mixture of spores of fungi 
known to colonise paints 
exposed to humid conditions 
for up to 12 weeks should show 
visual appearance of fungal 
growth. The treated sample 
should be free of it. 

Fungicide is used to treat 
paper goods to prevent 
mould growth in service. 

9 

Labels used on wine and beer 
bottles become degraded and 
stained by fungi and difficult to 
read when stored in cellars and 
cool stores. 

ASTM D 2020-03 

Samples of untreated material 
should demonstrate a high 
susceptibility to fungal growth 
in the test. Treated samples 
should be free of growth. 

9 

Biocide with fungicidal 
and bactericidal 
properties is used to 
protect PVC sheet 
materials from spoilage 
and degradation in 
service 

PVC sheet flooring used on 
solid floors can become 
colonised by bacteria and fungi 
on its under surface. This 
causes staining, cracking and 
detachment from the 
substrate. 

ISO 846 Parts A and C. 

Samples of untreated material 
should support bacterial and 
fungal growth. Treated material 
should be free of growth. 

inhibition 

Growth 
of 
moulds  occurring  on  the 
plasters  and  walling  in 
building structures 

10 

Surfaces  of  walls  exposed  to 
weather  can  be  infected  by 
saprophytic moulds. 

Field  tests  :  moulds  growth 
should  be  shown  on  untreated 
Treated  material 
material. 
should  be 
free  of  moulds 
growth. 

 
 
 
 
 
   
 
 
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5.5.6  PT6 Preservatives for products during storage 

In-can  preservatives  are  included  in  many  manufactured  products,  including  paints, 
adhesives and binders. They are used to control micro-organisms that may be present in the 
product  and  which  may  cause  deterioration  prior  to  use.  They  therefore  help  to  ensure 
product  integrity  during  normal  shelf  life.  Note:  Food  preservatives  and  cosmetics 
preservatives, which are used exclusively for this purpose, are not included in Product Type 
6. 

In  order  to  grow  in  a  manufactured  product,  a  micro-organism  must  have  access  to  both 
moisture (water) and a nutrient source. An extremely wide range of substances can act as a 
source  of  nutrition.  These  substances  may  be  utilised  by  micro-organisms  as  they  are,  or 
following some form of conversion or degradation. 

Utilisation  of  nutrition  sources  by  micro-organisms  results  in  the  loss  from  the  product  of 
one or more components, leading to reduced integrity and spoilage. By-products of microbial 
growth also contribute to spoilage.  Thus vulnerable products require an in-can preservative 
content for protection during the wet state, prior to use. 

The broad group of wet-state preservatives for the purpose of storage prior to use has been 
divided into the sub-categories and sub-scenarios: 

PT6.1 

Washing and cleaning fluids and human hygienic products 

6.1.1 Washing and cleaning fluids (human hygienic products) 

6.1.2 Washing and cleaning fluids (general) and other detergents 

PT6.2 

PT6.3 

Paints and Coatings (PN) 

Fluids used in paper, textile and leather production (P) 

6.3.1 Fluids used in paper production (Bulk raw materials in storage) 

6.3.2 Fluids  used  in  textile  production  (Bulk  raw  materials  in  storage)6.3.3

Fluids used in leather production (Bulk raw materials in storage) 

PT6.4 

Metal working fluid 

6.4.1 Lubricants (P) 

6.4.2 Machine oils (P) 

PT6.5 

Fuel 

PT6.6.  

Glues and Adhesives 

PT6.7 

Mineral slurries and other matrices 

Each  of  these  sub-scenarios  can  be  tested  as  described  in  5.5.2  and  5.5.5.1.  This  can  be 
summarised as follows. 

•  A relevant matrix must be chosen according to the intended use. This matrix should 
be  selected  in  a  way  that  it  can  easily  support  growth  if  no  biocide  is  present.  A 
reasonably  high  water  content  and  organic  matter  (either  from  the  matrix  itself  or 
added as a soiling agent) will allow for growth. 

• 

If  available,  a  standard  that  covers  the  matrix  must  be  chosen  (e.g.  for  glues  you 
might choose ASTM standard D 4783). From this test protocol the test organisms, the 
method  of  cultivating  the  test  organisms,  duration  of  the  incubation,  incubation 

 
   
 
 
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temperature, etc. can be extracted and integrated into a test protocol that follows the 
principles outlined above (e.g. by reducing the size of the inoculum). 

Examples  for  test  protocols  18that  follow  these  principles  are  listed  below.  Other  test 
methods  which  are  commonly  used  for  PT  6  can  be  found  in  Appendix  8.  However,  please 
note  that  these  test  methods  are  not  necessarily  appropriate  to  use;  they  are  listed  with 
comments  to  give  an  orientation  for  the  assessor  when  and  where  these  tests  can  be 
meaningful to prove a claim and when they aren’t: 

i. 

ii. 

iii. 

A  Method  for  Determining  the  Basic  Efficacy  of  Biocidal  Active  Substances  used  in 
Polymer Dispersions, IBRG PDG 16-001; 

A  Method  for  Determining  the  Basic  Efficacy  of  Biocidal  Active  Substances  used  in 
Aqueous-Based Paints, (IBRG2 P 16-001; 

Tier  1  Basic  Efficacy  Method  for  Biocidal  Active  Substances  used  to  Preserve 
Aqueous-Based Products, (IBRG2, IBRG PDG 16-007. 

These  documents  describe  methods  for  determining  the  basic  efficacy  of  biocidal  active 
substances in  an aqueous based matrix and are intended  for the generation  of tier 1 data. 
The  impact  of  additional  factors  like  temperature  and  chemical  stability  etc.,  depending  on 
the claim, would need to be tested. 

When a claim of an active is to reduce bacterial growth, all 3 methods work according to the 
same  principles,  but  differ  in  the  bacteria  used  as  they  are  specific  to  the  matrix  and  the 
strength  of  the  inoculum  (also  refer  to  5.5.4.1).  When  the  active  substance  also  claims  to 
reduce  fungal  growth,  it  will  be  necessary  to  differentiate  between  unicellular  yeasts  and 
filamentous  fungi  as  yeasts  can  be  counted  as  colony  forming  units,  whereas  filamentous 
fungi cannot (also refer to 5.5.4.2). 

The filamentous fungus Geotrichum candidum is an organism that forms filamentous chains 
of  fragmented  cells.  These  are  special  in  so  far  as  they  disintegrate  easily  into  single 
arthrospores. Enumeration of growth of this fungus can therefore be performed in the same 
way as for unicellular yeasts. Details for culturing this fungus are given in method ii (Paints). 

Whereas  methods  i)  deal  with  polymer  dispersions  and  ii)  deal  with  paints,  the  efficacy  of 
preservatives  in  all  other  matrices  in  PT  6  are  at  this  point  tested  according  to  a  generic 
method  shown  under  iii)  above.  It  provides  a  unified  approach  and  is  for  use  with  those 
materials  that  do  not  (yet)  have  a  specific  method  available  (e.g.  surfactants,  cleaning 
products, mineral slurries etc.). It is designed to satisfy the basic requirements described in 
this document. As with the above tests, it is based on a challenge test (multiple inoculations 
at weekly intervals) and has the same basic requirements. 

5.5.7 PT  7  Film  preservatives  and  PT  9  Fibre,  rubber  and  polymerised 
materials preservatives  

Uses  within  PT  7  (film  preservatives)  and  PT  9  (fiber,  leather,  rubber  and  polymerized 
material  preservatives)  often  overlap.  Sometimes,  PT  7  and  9  differ  only  in  the  manner  of 
application:  the  biocide  can  be  applied  as  a  coating  layer  onto  the  material  or  it  can  be 
incorporated into the material. Thus, the described requirements and principles apply in the 
same way to both PTs. 

18 IBRG website for test protocols: http://ibrg.org/Methods.aspx 

 
 
 
 
 
   
 
 
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When  selecting  the  appropriate  method,  consideration  must  be  given  to  the  release  mode 
characteristics  of  a  particular  biocide/material  combination.  Some  biocides  have  a  very  low 
solubility  in  water  and  hence  are  emitted  at  a  very  low  rate  from  a  matrix.  This  may  be 
sufficient  to  protect  a  material  that  is  inherently  highly  susceptible  and  which  micro-
organisms  may  penetrate  and  colonise.  However,  if  a  test  (e.g.  ISO  16869)  relies  on  the 
emission  of  the  biocide  from  the  matrix  into  an  agar  layer  to  measure  the  effect,  the  test 
would indicate that such a biocide has no function. Other materials, which are damaged by 
growth  on  their  surface  (especially  where  soiling  is  present)  due  to  the  production  of 
extracellular enzymes, may fail to be protected by a biocide with such a low emission rate. 
Thus, the choice of method will be highly dependent on the characteristics of the material as 
well as the biocide. The applicant should justify this for the product under evaluation. 

5.5.7.1 Simulation Tests (Tier 1 testing) 

The ideal test method  would present a material to a consortium  of  relevant test organisms 
under  conditions  that  simulate  real  life  realistically.  This  would  produce  effects  that  are 
identical to those observed in practice and allow a treatment to be identified with precision. 
There are methods that come closer to this ideal than others. For example, BS 3900 Part G6 
(Appendix 6) exposes painted panels that have been inoculated with a mixture of spores of 
fungi  known  to  colonise  paints  to  humid  conditions,  free  of  external  nutrients  (although 
these can be added with the inoculum if necessary) for up to 12 weeks (see Figure  8). The 
resulting growth on untreated coatings has a visual appearance very similar to that observed 
in  practice.  For  Tier  2  pre-exposure,  leaching  or  artificial  weathering  can  be  used  to  help 
explore service life. A comparison can be made between treated and untreated variants of a 
formulation.  A  similar  test,  that  forms  the  basis  of  many  of  the  military  standards  and 
specifications, is BS EN 60068-2-10:2005 (see Appendix 6); this test is applicable to a wider 
range of materials. Again, samples are inoculated and incubated under  conditions intended 
to simulate real life or at least be optimal for fungal growth. 

 
   
 
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Figure 8: Example of a Simulated Growth Test 

BS 3900 Part G6, Method Overview:  

Replicate sub-samples of both treated and untreated variants of each 
coating are sprayed with a suspension of spores of a range of fungi known 
to colonise surface coatings. The samples are then transferred to a humid 
chamber and incubated for up to 12 weeks.  The extent of growth is 
assessed using a rating scale and this, as well as photographs of the panels, 
are presented as the results.  

Rating scale: 0 = no growth, 1 = trace to 1% cover, 2 = 1 - 10% cover, 3 = 
10 - 30% cover, 4 = 30 - 70% cover and 5 = > 70% cover 

There is no pass/fail criterion in 
the standard but many workers 
in the coatings industry 
consider that growth 
represented by a rating of 2 is 
the maximum that would 
normally be tolerated.  An 
example of growth on an 
untreated coating is shown on 
the left. 

Example for growth level 5. 

Modifications of these methods have been made to allow them to study the effects on algae 
(the  IBRG  algal  test  method  for  surface  coatings)  and,  less  commonly,  bacteria. 
Effectiveness  is  assessed  in  these  tests  by  visual  appearance,  measuring  loss  of  weight  or 
determining changes in the physical properties of the material (e.g. resistance to bending or 
extension  under  load).  As  with  all  biological  tests,  some  degree  of  replication  will  be 
essential  and  tests  should  employ,  as  a  minimum,  three  replicate  sub-samples  of  each 
variant. Simulation tests are indeed very useful and provide valuable information especially 
for  specific  material/biocide  combinations  and  can  be  correlated  in  some  cases  to  service 
expectations. However, they can take a long time to perform and, in many cases, need to be 
adapted in some manner to accommodate a specific material. 

5.5.7.2 Tests based on artificial growth media (Tier 1 testing) 

By far the most commonly used methods for studying the performance of biocides intended 
to  protect  materials  are  those  based  on  artificial  growth  media  such  as  agar  plates.  For 
example, both ISO 846: 1997 and ASTM G21-09 are used widely in the plastics industry to 
measure  the  performance  of  fungicides  in  formulations  (also  ISO  16869:  2008).  ISO 846 
allows  for  studies  into  the  susceptibility  of  plastic  formulations  to  fungal  and  bacterial 
deterioration  by  attempting  to  make  the  plastic  the  sole  source  of  nutrients  for  the 
organisms  used,  as  well  as  providing  a  variant  that  provides  an  external  source.  It  also 
includes  a  service  life  simulation  test  variant  in  which  samples  are  buried  in  soil  and  then 
examined  for  loss  of  weight  and  strength  (extremely  useful  in  industries  manufacturing 
pipes and cables). Although making the plastic the sole source of nutrients might seem like 
the ideal way to examine the ability of a biocide to protect the material, in many instances it 
is the presence of soiling that leads to colonisation and subsequent damage to the polymer 
(sometimes  referred  to  as  bio-corrosion).  Thus,  for  certain  polymers,  the  presence  of 

 
 
 
 
 
   
 
 
 
 
 
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external nutrients is essential in determining the efficacy of a biocide. In many instances  a 
consortium  of  organisms  is  required  to  effect  colonisation  and  deterioration  of  the  material 
and, in general, methods that employ consortia should be selected. 

Similar  testing  technologies  as  those  used  for  plastics  exist  for  certain  textiles,  paper  and 
surface  coatings. The  most  commonly used are listed in Appendix 9; However, please note 
that  these  test  methods  are  not  necessarily  appropriate  to  use;  they  are  listed  with 
comments  to  give  an  orientation  for  the  assessor  when  and  where  these  tests  can  be 
meaningful  to  prove  a  claim  and  when  they  aren’t.  A  description  of  the  basic  principles  of 
tests on artificial growth media is given in Figure 9 using ASTM G21 as an example. 

The huge disadvantage of agar-plate based tests is the interference  of the growth medium 
with the biocide. The biocide can diffuse into the agar, demonstrating an effect there but at 
the  same  time  be  diluted  in  the  original  matrix.  A  less  soluble  substance,  which  does  not 
diffuse  into  the  agar,  may  in  contrast  show  a  false  negative  effect.  For  these  reasons,  a 
simulation test is always to be preferred over an agar-plate based test. 

Figure 9: An Example of an Agar Plate Based Test 

ASTM G21, Method Outline: 

are 

Replicate  samples  of  both  treated 
and 
are 
untreated  material 
embedded  in  a  mineral  salts-based 
agar  medium.  The  sample  and 
surrounding 
then 
agar 
inoculated  with  the  spores  of  a 
mixture  of  fungal  species  known  to 
colonise  plastics.  The  plates  are 
then placed into chambers in which 
the  humidity  is  maintained  at  > 
85%  RH  for  up  to  28  days.  The 
samples  are  then  inspected  for  the 
presence  of  fungal  growth.  Typical 
growth  on  an  untreated  material  is 
shown in the plate on the left. 

Growth on Untreated Plastic 

5.5.7.3 Tier 2 Testing 

Depending on the intended use, pre-exposure, leaching or artificial weathering can be used 
to  help  explore  service  life.  The  relevance  of  the  chosen  parameters  should  be  explained. 
There are no special tests or designs available for tier 2 testing. Basically, the same methods 
as in tier 1 can be applied except that the tested material undergoes pre-treatment. In some 
cases,  ageing  norms  can  be  employed  (e.g.  adaptations  of  EN  73:201419,  EN  84:199720, 
which are both developed for treated wood). In other cases, variations of the tier 1 methods 
can  be  used  (as  for  example  the  soil  burial  variant  of  ISO 846  as  described  above).  It  is 

19 Accelerated ageing test of treated wood prior to biological testing. Evaporative ageing procedure  
20 Accelerated ageing tests of treated wood prior to biological testing. Leaching procedure  

 
   
 
 
 
 
 
 
 
 
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particularly  important  to  show  growth  or  damage  on  the  untreated  material  under  service-
life conditions. 

In some cases it may not be necessary to use an artificial inoculum for tier 2 tests. It may 
be  possible  to  use  a  test  medium  colonised  naturally  so  that  it  is  representative  of  the 
organisms  that  are  typically  encountered  during  the  use  of  the  product.  It  may  be  valid  to 
use  lower  levels  of  contamination  such  as  those  encountered  in  practice.  In  some  cases 
there  may  be  a  need  to  include  application-related  test-organisms  in  addition  to  standard 
test-organisms.  In  any  case,  the  applicant  should  provide  a  rationale  as  to  why  the  test 
organisms are relevant for the respective application/s of the preservative. Representatives 
for all claimed organisms should be tested. 

5.5.7.4 Tier 3 Testing 

In some cases, tier 3 testing might be needed to support specific claims. These can be field 
tests  where  treated  materials  are  compared  to  untreated  materials  in  use.  For  example, 
treated house facades could be compared to untreated house facades in the same area and 
the time until re-painting is needed could be measured. Likewise, the replacement time for 
untreated buried cables compared to treated ones can be studied in a field test. Care has to 
be taken that the conditions for the treated and untreated materials are the same or at least 
comparable and that other parameters than the parameters observed are not influencing the 
results. The validity of the conclusions may need to be reinforced by statistical analysis etc., 
especially if any differences observed are small. 

Table 12: Basic Requirements for a Valid Test Protection 

The following summary provides a guide to the basic requirements for a valid test: 

i. 

ii. 

iii. 

iv. 

v. 

vi. 

vii. 

A relevant model matrix should be chosen to represent the material(s) which 
must be protected; 
Relevant use conditions should be chosen in terms of humidity temperature and 
soiling; 
An untreated variant of the test material must be included and show the pattern 
of growth/deterioration that the biocide is intended to prevent at the end of the 
test; 
The test should employ organisms that are relevant to the material/problem 
being addressed; 
Tests that employ a consortium of organisms should be favoured over those that 
use single species; 
A minimum of three replicate test pieces of both treated and untreated materials 
should be employed; 
The final data should include either some indication of the impact of service 
conditions on the performance of the treated material/article or data from an 
ageing. 

5.5.7.5 Prevention of Odour by odour-producing micro-organisms 

With  most  of  the  biocidal  functions  within  PT  7  and  9,  test  conditions  simulate  in-use 
conditions  rather  well and the effects of microbial growth or activity  can be  observed quite 
easily.  With  the  control  of  odour,  this  is  much  harder  to  achieve  in  a  laboratory  test,  as 
odour often cannot be measured in a simple manner. 

 
 
 
 
 
   
 
 
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Laboratory tests to simulate odour production are currently not available, though some work 
is done to develop such tests (for example a test to inhibit the bioconversion of L-leucine to 
iso-valeric  acid,  representing  a  dominant  compound  of  foot-odour).  Thus,  at  present,  the 
prevention of odour is in most cases measured indirectly by measuring microbial inhibition. 

There are two major types of test that have traditionally been used with textiles (and related 
materials).  The  first  major  group  employs  agar  plates  and  the  other  major  group  uses 
suspension  in  an  aqueous  medium.  In  both  cases,  the  impact  of  a  treated  textile  on 
populations of (usually) bacteria are studied. An overview is given in Appendix  10; however 
please  note  that  the  test  methods  listed  are  not  necessarily  appropriate  to  use;  they  are 
listed with comments to give an orientation for the assessor when and where these tests can 
be meaningful to prove a claim and when they aren’t. 

Agar plate-based tests 
Agar  plate-based  tests  are  not  recommended.  These  tests  have  almost  no  useful  utility  in 
measuring  effects  intended  to  control  odour  in  textiles.  Such  tests  rely  on  the  biocide 
migrating  from  the  textile  into  the  agar  medium  at  sufficient  concentration  to  inhibit  the 
growth of bacteria either seeded into the agar or placed onto it (see Figure 9). The diffusion 
characteristics vary hugely from one biocide to another and from one textile to another and 
the growth medium itself presents a large soiling load to be overcome by the biocide. Larger 
areas  clear  of  growth  are  often  associated  with  more  potent  effects  but  they  could  be 
attributed equally to differences in the leaching rate of a biocide from a material. 

Suspension tests  
The second major group, the suspension tests, measure changes in the size of a population 
following contact with a treated textile. A number of protocols are described in Appendix 10. 
However, most employ relatively high concentrations of nutrients in the suspending medium 
so  that  their  application,  like  the  agar  diffusion  methods,  can  lead  to  over-treatment  of 
textiles. Thus, these methods should not be used. By using lower concentrations of nutrients 
in the suspending medium and using pre-treatments such as laundering, these methods can 
be  adapted  for  use  in  measuring  effects  on  odour.  Such  an  adaptation  has  been  applied  in 
the  OECD  Tier  1  method  for  treated  articles  (porous  materials21)  and  the  IBRG  Textile 
Method22. These are described schematically in Figure 10 and are based on the ‘germ’ count 
or absorption phase  of  ISO 20743: 2007 where the amount of nutrients present in the  cell 
suspension has been reduced substantially. 

Many treated materials would certainly be capable of demonstrating activity in a suspension 
test.  Activity  against  a  consortium  of  bacteria  (e.g.  against  a  range  of  Gram  Positive  and 
Gram  Negative  bacterial  species  such  as  Staphylococcus  epidermidis,  Corynebacterium 
xerosis,  Proteus  vulgaris,  Escherichia  coli,  etc.)  would  probably  inhibit  the  production  of 
odour.  However,  excess  exposure  of  the  skin  of  the  wearer  should  be  minimized  as  far  as 
possible. Therefore, tests adapted to textile treatments such as the OECD Tier 1 method and 
the IBRG Textile method (Figure 10) are preferable. 

21  OECD  (OECD  ENV/JM/MONO(2014)18:  Guidance  Document  for  Quantitative  Method  for  Evaluating 
Antibacterial  Activity  of  Porous  and  Non-Porous  Antibacterial  Treated  Materials  (OECD  Series  on 
Testing and Assessment No. 202 and Series on Biocides No. 8). 

22  IBRG,  International  Biodeterioration  Research  Group  (2013):  Quantitative  Method  for  Evaluating 
Bactericidal Activity of Textiles and Porous Materials and Articles. IBRG TEX/13/005 (www.ibrg.org). 

 
   
 
 
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Figure 10: OECD/IBRG Tier 1 Textile Test 

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Tier 2 testing  
In many cases, a large fraction of the active substance incorporated in a textile is lost during 
laundering, either through emission of loosely or only partially bound material or associated 
with loss of fibres (lint). This also means that there is potential for active substances to be 
transferred  from  treated  materials  to  non-treated  materials  when  laundered  together.  In 
general, the  emission  rate is  rarely continuous either to the  environment or to the  wearer. 
Moreover,  other  chemicals  from  the  textile  treatment  as  well  as  chemicals  used  in  the 
laundering process might interfere with the function of the biocide. 

In general, the effects required to prevent the formation of odour in shoes and apparel are 
subtle.  The  greatest  demand  on  them  is  usually  in  maintaining  activity  following  multiple 
laundering  cycles.  Therefore,  simulation  of  service  life  conditions  by  laundering  and  ageing 
are  essential.  Care  must  be  taken  to  maintain  the  functionality  and  to  minimise  excess 
exposure of the environment through emissions of the biocide in use, during cleaning and at 
the  time  of  disposal.  The  method  described  in  Figure  10  (as  well  as  chemical  analysis)  in 
combination with laundering  cycles can be useful in measuring the maintenance  of efficacy 
in service. 

An active substance or a biocidal product is often intended to treat a wide range and mix of 
textile  types  with  a  wide  variety  of  anticipated  demands  and  expectations  of  durability.  It 
might  be  difficult  to  address  every  potential  combination  and  garment  type.  However, 
studies  on  typical  textile  blends  could  be  used  to  provide  appropriate  efficacy.  Some 
examples are given in Table 13 below. 

 
 
 
 
 
   
 
 
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Tier 3 testing  
At  present  the  only  truly  reliable  methods  for  demonstrating  anti-odour  functionality  is 
through  replicated  and  statistically  designed  wearing  trials.  Tier  1  and  2  tests  described 
above  can  provide  useful  data  related  to  durability  etc.  but  care  must  be  taken  when 
interpreting the data they produce. For example, a treatment may be applied to only certain 
parts of a garment or shoe or it may be present on only a certain number of filaments in the 
weave  of  a  textile.  In  the  bioassay,  the  inoculum  is  dispersed  throughout  the  whole  of  the 
sub-sample  of  textile  and  any  active  substance  released  would  be  able  to  migrate 
throughout  that  inoculum  whereas  in  use,  this  may  not  occur.  The  humidity  produced  by 
bodily  excretions  might  trigger  less  release  of  the  biocide  than  the  liquid  suspension  the 
textile  is  covered  with  in  the  test.  The  bacterial  populations  present  on  the  skin  might  be 
less affected by the biocide as compared to the testing consortium employed. Consequently, 
user trials are proposed as reliable methods to prove anti-odour effects, especially in case of 
textiles,  but  also  suitable  microbiological  studies  with  relevant  odour-causing  micro-
organisms  can  be  acceptable  ways  to  prove  anti-odour  claims.  A  standard  with  human 
assessors which could possibly be adapted to test anti-odour claims is EN 13725. 

Table 13: Odour: Example Claims, Problems and Testing Approaches 

Claim 

PT  Proof Required 

Example Method 

Carpet is treated to 
prevent odours caused 
by mould growth. 

9 

Data should show that the 
treated carpet does not 
support fungal growth 
whereas the untreated one 
does. 

A method such as AATCC 174 can 
be used to demonstrate resistance 
to fungal growth. For active 
substances that do not migrate 
from the fibres/backing a cabinet-
based simulation test may be more 
appropriate. 

The effect should be shown to 
be sufficiently durable. 

Activity should be shown to persist 
following simulated ageing. 

A sports vest is 
treated to inhibit the 
production of odour. 

9 

Data from a field trial should 
show that odour is reduced in 
treated sports shirts when 
compared with untreated 
ones. 

The effect should be shown to 
be of sufficient durability 
during service life to match 
any claim made. 

Wearing trial or scientifically valid 
odour based simulation study. 

A comparison of the effectiveness 
both before and after simulated 
ageing/washing should be 
performed. This could be performed 
either through field trials, 
simulation tests or the use of a test 
such as the OECD Tier 1 method. 
The latter could be used to 
demonstrate that sufficient activity 
is still present after washing/ageing 
to elicit an antimicrobial effect. 

 
   
 
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5.5.8 PT8 Wood preservatives  

General Introduction 

This document deals with the evaluation methodology of efficacy tests for wood 
preservatives biocidal products that are applicable in the frame of the EU Biocidal Products 
Regulations (BPR) for the authorisation of biocidal products (BPR Annex VI). 

The document is not intended to replace standards, standardized methods or other methods 
used as reference for developing the required data. It is considered as scientific guidance 
and the reader is advised to refer to the standards themselves or appropriate literature in 
case details should require further clarification. 

The aim of this document is to provide a common base for the assessment of the efficacy for 
the biocidal product authorization for PT8 products for the applicants and the Competent 
Authorities (CAs). 

Although alternative test methods could be taken into account, this document is mainly 
based on the EN 599-1 standard for preventive uses and on the EN 14128 standard for 
curative uses. 

This document covers the products used for the preventive treatments of wood (including 
the saw-mill stage), by the control of wood-destroying or wood-disfiguring organisms 
(temporary treatments of logs in the sawmill or log yards, temporary treatments of green 
sawn timber, treatments of sawn timber including round timber, treatments of wood based 
panel) and products used for the curative treatments of sawn timber in service. 

For product already on the market before entering into force of the standards (in 1990 for 
EN 599 and in 2004 for EN 14128): 

•  Efficacy data on the product should be provided. 

•  The assessment of the product efficacy should be based on expert judgement; 

•  Some data taken from the literature or used in certification could be accepted on 

case by case basis. 

When the data are not enough robust to demonstrate the efficacy of the product, new tests 
according to EN 599 and/or EN 14128 will be required. 

At the review time of this document, it has been chosen to include the catalogue of uses in 
the Chapter 7 of the Technical notes for guidance (TNsG) on product evaluation (PT8). The 
inclusion of the catalogue of uses to this document is to provide a common basis to 
harmonize the claims of the product. It will facilitate in a second time the mutual recognition 
by listing the elements of the claim in the same order and using the same terminology. On 
the label, the categories related to the product should be presented as described in the 
following paragraphs. The codes increase the readability of this document and are not 
expected on the label. 

Concerning the updating of this document, it should be considered as a living document and 
will be reviewed on a regular basis and updated if necessary, under ECHA’s procedures. 

The tests should be performed according to the current version in force of this document. 
Any tests initiated before the endorsement of the new version remain acceptable.  

5.5.8.1 Label claims 

In order to harmonize the efficacy issues, it is proposed that the different uses of the 
product are presented following the proposal below. This should follow the order of the 
categories listed below. 

 
 
 
 
 
   
 
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The aim of this categorisation is to have an explicit answer on the following questions: 

•  Where is the product used? 

•  What is the product used for? 

•  How is the product used? To control which organisms? 

The data which support the efficacy should also follow this format. 

The main categories that should be present on the label are listed in Table 14 and are 
detailed in the following paragraphs. 

Table 14: Different categories and the related product codes 

Categories 

User category 

Wood category 

Wood product 

Code for product 

A.xx 

B.xx 

C.xx 

Application aim & Field of use 

D.xx & E.xx 

Method of application and rate 

Target organisms 

F.xx 

G.xx 

5.5.8.1.1 User Category (Code for Product A.xx) 

Information on the intended users of the product has to be presented on the label, the 
different user categories are presented in Table 15. 

Table 15: User categories 

User Category 

Example 

Product Code 

Non-professional/general public 

Product used at home by consumers 

A.10 

Industrial 

Professional 

Industrial applicator  

Pest control operator 

A.20 

A.30 

 
   
 
 
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5.5.8.1.2 Wood Category (Code for product B.xx) 

This section deals with the wood category and not the use classes as defined in EN 335 
standard. From an efficacy point of view, in EN 599-1, annex D the wood timbers are divided 
into two categories: softwood and hardwood. 

Softwood and hardwood species of timber react differently to the degree and the type of 
attack by certain biological agents. 

In most cases, the tests are performed with softwood. In some cases it is acceptable for this 
data to be read across to hardwoods, but in other cases specific testing against hardwoods is 
required. (see EN 599-1). 

Table 16: Wood categories 

Wood Category 

Softwood 

Hardwood 

Product Code 

B.10 

B.20 

5.5.8.1.3 Wood Product (Code for product C.xx) 

Table 17 below describes the types of wood products that are used as building materials or 
in  the  manufacture  of  furniture.  Wood  products  are  divided  in  two  main  categories:  solid 
wood and wood based panels. Based on European standards, wood based panels are divided 
in four categories: plywood (EN 636), OSB (EN 300), Particles (EN 309 & EN 312) and Fibers 
(EN 622). 

Table 17: Wood product categories 

Wood Category 

Solid wood 

Reconstituted solid wood 
Engineered solid wood products produced by processes involving pressure, adhesives and 
binders 

Panels 

Plywood panels 

OSB panels 

Particles panels 

Fibers panels 

Product Code 

C.10 

C.11 

C.20 

C.21 

C.22 

C.23 

C.24 

5.5.8.1.4 Application aim and field of use 

5.5.8.1.4.1 Application aim (code for product D.xx) 

A preventive treatment is used to prevent sound wood from being infected by wood 
destroying agents and/or disfiguring fungi. The curative treatment is used to kill infective 
organisms that have already attacked the wood, to prevent them from spreading in the rest 
of the wood. 

 
 
 
 
 
   
 
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The preventive treatments are most of the time used during the manufacturing process but 
can also be done when the wood is in its service situation (e.g. framework of the building, a 
bridge.). 

According to the fact that a product can be used in wood preventive treatments, in curative 
treatments and sometimes both, and according to the fact that wood preservative and 
curative treatments are not covered by the same treatments, it is proposed to split the 
application aims as presented in Table 18. 

The aim of this classification is to ensure having the same classification throughout the EU. 

Table 18: Application aim 

Application Aim 

Kind of Treatment 

Product Code 

Preventive 

Temporary preventive treatment / logs 

D.10 

Temporary preventive treatment / green sawn timber 

D.20 

Preventive treatment / blue stain in service 

Preventive treatment-use class (cf. the following 
section for the field of use – code E) 

Curative  

Curative treatment / wood in service 

Preventive  

Other (for e.g. pole maintenance) 

D.30 

D.40 

D.50 

D.60 

5.5.8.1.4.2 Field Of uses (Code For Product E.xx) 

The use classes described in EN 335:2013 are defined in terms of service conditions, with 
reference to the generalised moisture content and the prevailing biological agents of 
deterioration. The different classes (and their related application codes) are presented in 
Table 19. 

•  Use class 1: situation in which the wood or wood based product is inside a 

construction, not exposed to the weather and wetting; 

•  Use class 2: situation in which the wood or wood-based product is under cover and 
not exposed to the weather (particularly rain and driven rain) but where occasional, 
but not persistent, wetting can occur; 

•  Use class 3: situation in which the wood or wood-based product is above ground and 

exposed to the weather (particularly rain); 

•  Use class 4: situation in which the wood or wood-based product is in direct contact 

with ground or fresh water; 

•  Use class 5: situation in which the wood or wood based product is permanently or 

regularly submerged in salt water (i.e. sea water and brackish water). 

Use class 3 is split into two sub-classes: 

•  3.1: wood and wood based products will not remain wet for long periods. Water will 

not accumulate; 

•  3.2: wood and wood-based products will remain wet for long periods. Water may 

accumulate. 

 
   
 
 
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The use classes 4.1 and 4.2 described in the former version of the EN 335 standard (2009) 
have been merged into a single use class 4, including both wood in exterior, in ground 
and/or fresh water contact. 

Table 19: Different fields of uses 

Field of Uses 

Use class 1 

Use class 2 

Use class 3* 

Use class 3.1 

Use class 3.2 

Use class 4 

Use class 5 

Product Code  

E.10 

E.20 

E30 

E.31 

E.32 

E.40 

E.50 

* includes use class 3.1 and use class 3.2 

5.5.8.1.5 Method of application and application rate (Code for product F.xx): 

The various methods available can be broadly split into three groups: 

•  Superficial treatments: Such non-pressure processes include brush, spray, roller, 

pad application and immersion (dipping) processes (where the wood can be in 
contact for preservative for periods of time ranging from a few minutes to several 
hours). The application rates are commonly expressed in g/m2, ml/m2. 

•  Penetrating treatments: Such processes include the vacuum pressure, alternating 
oscillating pressure, double vacuum and non-pressure processes such as diffusion 
treatments. The application rates are commonly expressed in kg/m3. 

•  Other treatment methods: For application methods different from those described 

above (fumigation, injection), either specifically relevant data or some justification for 
non-inclusion of data (i.e. details on penetrability/retention, etc.) will need to be 
provided to the CA for consideration. 

Some  PT  8  products  are  designed  to  be  used  with  a  top  coat,  e.g.  primers  for  window 
framing. If a top coat is needed according to the manufacturer, this must be applied with the 
product.  When  a  more  general  use  is  envisaged,  generic  coating  materials  can  be  used 
according to the norms performed. 

Table 20: Method of application 

Method of application 

Product Code  

Superficial application / brush/roller/pad treatment  

Superficial application / spray treatment  

Superficial application / flow coat /aspersion  

Superficial application / foam treatment  

F.10 

F.11 

F.12 

F.13 

 
 
 
 
 
   
 
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Method of application 

Product Code  

Superficial application / dipping treatment  

Injection  

Pressure process 

Pressure process / vacuum pressure impregnation 

Pressure process / double vacuum 

Fumigation 

Fumigation bubble 

Pole in services fumigation 

Mixing with glue and mortar 

Diffusion 

Solid pellets / rods 

Pole bandage / wrapping / pad application 

Other application methods 

F.14 

F.20 

F.30 

F.31 

F.32 

F.40 

F.41 

F.42 

F.50 

F.60 

F.61 

F.62 

F.70 

5.5.8.1.6 Target organisms (Code for product G.xx) 

This  section  describes  the  main  categories  of  target  organisms,  in  relation  to  the  claimed 
uses  of  the  product,  either  for  treatments  to  prevent  biological  attack,  or  for  curative 
treatments to disinfest or to eradicate existing attack. 

Appendix 11 gives more information on the principle target organisms. 

There are a number of possible effects on target organisms resulting from the proposed use 
of a wood preservative product. The efficacy data for a wood preservative must be suitable 
to  demonstrate  the  efficacy  of  products  applied  as  either  pre-treatments  to  prevent 
biological attack, or as curative treatments to disinfest or to eradicate existing attack. These 
may  be  in  a  variety  of  forms;  they  may  yield  toxic  values,  mortality  values,  subjectively 
derived ratings or effective retention values. 

On  the  claimed  matrix,  the  target  organisms  against  which  an  efficacy  is  claimed  must  be 
clearly described. For the purpose of harmonisation, it is proposed that the target organism 
presented  in  Table  21  should  be  used,  although  these  should  not  be  considered  as  an 
exhaustive  list.  The  species  presented  below  are  the  species  being  representative  of  wood 
attacking  organisms.  For  specific  claims,  efficacy  data  against  each  named  target  pest  will 
be required. 

 
   
 
 
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Table 21: Examples of target organisms for wood preservatives  

(N.B. these examples are not intended to be exhaustive with respect to target organisms or prescriptive with respect to data  to be 
generated). 

Target organisms 

Common English term 

Code F for 
product 

Target organisms 
according to EN 1001 

Classification 

Scientific name 

Fungi 

Wood rotting fungi 
Wood rotting 
basidiomycetes 
Soft rot fungi 

Wood  
discolouring fungi 

Insects 

Beetles 

Termites 

Wood destroying 
marine organisms 

G.10 
G.11 
G.12 
G.21.1 

G.21.2 

G.22 

G.30 
G.31 
G.32 
G.33 
G.40 
G.50 
G.51 

G.52 

G.53 

G.60 

G.61 
G.62 

Brown rot fungi 
White rot fungi 
Soft rot fungi 
Sapstain fungi (bluestain 
mainly) 
Bluestain in service 

Mould fungi 

Wood boring beetles 
House longhorn beetle 
Common furniture beetle 
Powder post beetles 
Fresh wood insect 
Termites (genus claimed) 
Subterranean termites (genus 
claimed) 
Drywood termites  
(genus claimed) 
Tree termites  
(genus claimed) 
Marine borers  
(genus claimed) 
Mussels 
Crustaceans 

Fungi 

Basidiomycetes 
Basidiomycetes 
Ascomycetes, Deuteromycetes 
Ascomycetes,  
Deuteromycetes 
Ascomycetes,  
Deuteromytcetes 
Ascomycetes,  
Deuteromycetes, 
Insecta 

Coleoptera 

Coleoptera 
Isoptera 

e.g. Gloeophyllum trabeum 
e.g. Coriolus versicolor 
e.g. Chaetomium globosum 
e.g. Ophiostoma piliferum 
(Ceratocystis pilifera) 

e.g. Aureobasidium pullulans 

e.g. Aspergillus niger 

e.g. Hylotrupes bajulus 
e.g. Anobium punctatum 
e.g. Lyctus brunneus 
e.g. Scolytus spp. 

e.g. Reticulitermes spp., 
Coptotermes spp. 

e.g. Cryptotermes spp. 

e.g. Nasutitermes spp. 

Teneridae, Pholadidae 
Isopoda, Amphipoda 

e.g. Toredo sp., Martesia sp. 
e.g. Limnoria spp., Chelura spp. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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5.5.8.1.7 Examples of a claimed matrix 

To illustrate the previous sections described, the following table gives an example of claimed 
matrix  based  on  the  categories  from  the  catalogue  of  uses.  This  framework  should  be 
followed  for  the  efficacy  claim’s  part  of  the  label.  Only  the  categories  and  the  matrix 
wordings (not the code) are expected to be listed on the label. 

This  matrix  allows  a  harmonisation  of  the  efficacy  elements  presented  in  the  dossier  for 
product authorization. Elements in the claimed matrix must be present on the physical label. 

Table 22: Examples of claim matrix based on the application codes for product 

Categories 

Matrix Wording 

Label 1 

Code 
Product 

for 

User category 

Industrial 

A.20 

Wood category 

softwood and hardwood 

B.10; B.20 

solid wood 

C.10 

preventive treatment - use class 3.2 

D.40; E.32 

Wood product 

Application aim 
and field of use 

Method of 
application and 
rate 

Target 
organisms 

superficial application/dipping treatment 
application rate: 100 g/m² in the analytical zone 
a top coat must be applied. 
pressure process/vacuum impregnation 
application rate: 50 kg/m3 in the analytical zone 

wood boring beetles 

termites (genus Reticulitermes) 

brown rot fungi 

white rot fungi 

F.14 

F.31 

G.30 

G40 

G.10 

G.11 

A.20 

Label 2 

User category 

Industrial 

Wood category 

softwood and hardwood 

B.10; B.20. 

Wood product 

Application aim 
and field of use 

Method of 
applicationand 
rate 

solid wood 

preventive treatment - use classes 2, 3 and 4 

C.10 

D.40 - E.20; 
E.30; E.40 

superficial application/dipping treatment 
application rate in the analytical zone: 
UC 2: 80 - 120 g/m² 
UC3 (coated): 100 – 160 g/m2 
pressure process/vacuum pressure impregnation 

F.14 

F.31 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
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application rate in the analytical zone: 
UC2: 30 kg/m3 
UC3: 40 - 70 kg/m3 
UC4 (softwood): 80 – 150 kg/m3 
UC4 (hardwood): 100 – 150 kg/m3 

brown rot fungi 

white rot fungi 

soft rot fungi 

wood boring beetles 

termites (genus Reticulitermes) 

Industrial 

softwood 

solid wood 

G.10 

G.11 

G.12 

G.30 

G.40 

A.20 

B.10. 

C.10 

temporary preventive treatment - use class 1 

D.20 E.10 

superficial application / dipping treatment 
application rate 100 g/m² in the analytical zone 

F.14 

sapstain 

mould fungi 

G.21.1 

G.22 

Target 
organisms 

Label 3 

User category 

Wood category 

Wood product 

Application aim 
and field of use 

Method of 
application and 
rate 

Target 
organisms 

5.5.8.2 Available data 

5.5.8.2.1 Standard test mthods 

When considering the overall evaluation of proposed claims, CAs should ensure that the test 
methods (data, method of application and application/dose rates used in the tests, product 
tested) are appropriate to demonstrate the efficacy claimed on the label for the product. 

Many standard protocols currently exist to test wood preservatives; the lists of standards for 
the efficacy assessment of wood preservatives are available on the ECHA Biocides Efficacy 
Working Group webpage [http://echa.europa.eu/about-us/who-we-are/biocidal-products-
committee/working-groups/efficacy]. . For PT8, the CEN standards are highly recommended. 

Two main categories of treatment are described: 

•  Preventive treatments, which are covered by EN 599-1; 

•  Curative treatments, which are covered by EN 14128. 

Some other treatments (C.20: green sawn timber) are covered by other standards (e.g. CEN 
TS 15082). 

It is highly recommended to perform the studies according to these standards. If the 
standards are not applicable or suitable, the applicant may adapt the methodology or use 

 
 
 
 
 
 
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another method (including his own method). When a standard is modified or when a non 
CEN standard is used, a robust justification and description have to be provided. For very 
specific cases, tests or ageing procedures could be waived with a robust justification. The 
study submitted has to provide a clear answer to the issue. 

In the general part of the TNsG on data requirements it is mentioned that the test (and the 
data generated) should be based on sound scientific principles and practices. Compliance 
with quality standards is highly recommended. 

In the TNsG on product evaluation, it is mentioned that for efficacy testing, the principles of 
Good Laboratory Practice (GLP) are not required by the legislation. However this guidance 
indicates that the spirit of such principles should be applied for the testing of efficacy. 

Particular attention should be paid to: 

•  what information is needed to substantiate a ‘claim matrix’; 

• 

• 

the Quality Assurance procedures which should be adopted (cf. ISO 17025 for testing 
and certification); 

the overall evaluation of the data package when the completeness and adequacy of 
the data are compared with the label claim. 

For products intended for application as solids, pastes or encapsulated forms and those 
intended for curative (in-situ) use, modification of the relevant protocols/testing strategies 
may be done or other direct evidence may be submitted on their potential efficacy against 
the claimed target organisms (e.g. for pastes such evidence could be in the form of 
penetrability and retention characteristics). 

The test methods used to provide data should be relevant to the target organisms and 
application processes claimed on the label (see EN 599-1 and individual test standards). 

It has to be noted that in some cases, a different formulation from which an authorization is 
sought could be tested. The results could be accepted by the RMS in a case by case 
approach (see section 5.5.8.3 of this guidance and Annex A of the EN 599-1 and EN 14128). 
A full composition of the tested product and a robust justification why the test is relevant 
should be provided. 

For EN113, where the protocol states that several organisms have to be tested in order to 
fulfil the efficacy criteria, it is recommended that all testing is done in the same laboratory at 
the same time. The sponsor must have the right to provide his rational for justification why 
the simultaneous testing may have not been followed. Derogation (inter alia) is acceptable 
i.e. in the following cases: 

•  where the test was performed with limited organisms and later completed with 
additional organisms which could be tested in another laboratory (extension of 
claim); 

•  where the laboratory cannot run the test with specific targets; 

•  where the laboratory has ceased to provide services; 

• 

in the case where a ‘simultaneous test’ is not available, but valid tests (according to 
the criteria in the standard) are available. 

Table 21 and Table 23 below are informative for the test methods used. The user should also 
refer to EN 599-1 or EN 14128 depending on the claims. 

 
 
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Table 23: Preventive treatments: List of available standards and others methods used in wood preservation 

Treatment of solid wood 

(List of standards mentioned in the tables 1 to 5 
of EN 599-1) 

Organisms 

Code  
for 
product 

Temporary 
treatment 
of logs 

Temporary 
treatment  

Note  1:  In  some  conditions,  ageing  tests  (EN  84,  EN  73)  or 
natural weathering are required (see EN 599-1) 

Note  2:  It  is  highly  recommended  to  refer  to  EN  599-1  to 
determine the tests to be done in accordance with table 1 to 5 
of EN 599-1 

Treatment of wood 
based panels23 

Brown rot 
fungi 

G.10 

White rot 
fungi 

G.11 

Soft rot 
fungi 

Sapstain 
fungi 

Bluestain 
fungi 

G.12 

G.21.1 

G.21.2 

Mould fungi 

G.22 

Wood boring 
beetles 

G.30 

Use Class 1 

Use 
Class 2 

Use 
Class 3 
EN 113 

EN 113 

EN 839 

EN 330 

EN 113 

EN 839 

EN 330 

Use 
Class 4 

Use 
Class 5 

EN 113 

EN 252 

EN 113 

EN 252 

ENV 807 

EN 252 

EN113 

ENV 12038 

EN113 

ENV 12038 

ENV 807 

No CEN 
standard* 

No CEN 
standard* 

No CEN 
standard* 

No CEN 
standard* 

EN 152 

EN 152 

EN 152 

EN 152 

EN 46 

EN 47 

EN 46 

EN 47 

No CEN 
standard 

EN 46 

EN 47 

EN 47 

EN 47 

EN 49-2 

EN 49-2 

EN 49-1 

EN 49-1 

EN 49-1 

EN 20-2 

EN 20-2 

23 For wood based panels, the reader is aware that standards can be adapted in specific cases (e.g. CEN/TS 15083-2 for soft rot fungi, EN 20-
2 for powder post-beetle and EN 117 and EN 118 for termites) 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Organisms 

Code  
for 
product 

Temporary 
treatment 
of logs 

Temporary 
treatment  

House 
longhorn 
beetle 

Common 
furniture 
beetle 

Powder 
post-beetle 

Fresh wood 
insect 

G.31 

G.32 

G.33 

G.40 

Termites 

G.50 

Marine 
borers 

G.60 

Blank cell: Not applicable;  

No CEN 
standard* 

Treatment of wood 
based panels23 

Treatment of solid wood 

(List of standards mentioned in the tables 1 to 5 
of EN 599-1) 

Note  1:  In  some  conditions,  ageing  tests  (EN  84,  EN  73)  or 
natural weathering are required (see EN 599-1) 

Note  2:  It  is  highly  recommended  to  refer  to  EN  599-1  to 
determine the tests to be done in accordance with table 1 to 5 
of EN 599-1 

Use Class 1 

EN 49-2 

Use 
Class 2 
EN 49-2 

Use 
Class 3 
EN 49-2 

Use 
Class 4 

Use 
Class 5 

EN 20-1 

EN 20-1 

EN 20-1 

EN 20-2 

EN 20-2 

EN 20-2 

EN 46 

EN 47 

EN 46 

EN 47 

EN 49-1 

EN 49-1 

EN 49-2 

EN 49-2 

EN 46 

EN 47 

EN 49-1 

EN 49-2 

EN 47 

EN 47 

EN 49-2 

EN 49-2 

EN 20-1 

EN 20-1 

EN 20-1 

EN 20-2 

EN 20-2 

EN 20-2 

EN 20-2 

EN 20-2 

EN 118 

EN 117 

EN 118 

EN 118 

EN 117 

EN 117 

EN 117 

EN 252 

EN 117 

EN 275 

* National standards available (see the ECHA Biocides Efficacy Working Group webpage [http://echa.europa.eu/about-us/who-we-
are/biocidal-products-committee/working-groups/efficacy]). 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Table 24: Curative treatments: List of available standards used in wood curative 
treatments (based on EN 14128) 

Organisms 

Code for Product 

Curative treatment 

Brown rot fungi 

White rot fungi 

Soft rot fungi 

Sapstain fungi 

Blue stain fungi 

Mould fungi 

Wood boring beetles 

House longhorn beetle 

Common furniture beetle 

Powder post beetles 

Fresh wood insect 

Termites (genus claimed) 

Marine borers (genus claimed) 

*Blank cell: Not applicable 

5.5.8.2.2 Preventive treatments 

G.10 

G.11 

G.12 

G.21.1 

G.21.2 

G.22 

G.30 

G.31 

G.32 

G.33 

G.40 

G.50 

G.60 

ENV 1390 

EN 48 or EN 370 

No CEN standard available 

No CEN standard available 

Most of the available data are laboratory generated and related to the organisms for which 
biocidal efficacy is claimed. 

Field tests, although desirable in cases where the product is intended for use in the more 
severe service environments (e.g. in ground contact (use class 3, 4 and 5)) are considered 
mandatory to fulfil the minimum performance criteria, according to the tests required in the 
paragraphs related to the use classes. As this could lead to a significant delay before a new 
product could be introduced to the market, literature, monitoring or other methods provided to 
support the derived application rate could be accepted in case by case by the CAs (see also 
notes in sections 5.5.8.2.2.3 and 5.5.8.2.2.4). 

The assessment of the preventive efficacy of wood preservative formulations has to be made 
from values derived from a relevant biological test. These values are either the actual 
quantitative amounts of the product established in the test as causing the appropriate level of 
mortality of the target organism, or they represent the threshold limits, the so-called 'toxic 
values'. These toxic values are two concentrations in the series used in the test, the first which 
just permits continued attack and the second which just prevents it. 

5.5.8.2.2.1 Temporary treatments of logs (in the sawmill or in storage area) 

This kind of treatment is used to prevent the degradation of logs which do not immediately 
have their bark removed. Indeed, some microscopic fungi (e.g. stain) infect the wood and/or 
some species of insects belonging to the family of Scolytidae and Bostrychidae (named “Fresh 
wood insect” in Table 21: ) lay their eggs between the bark and the wood. 

To prevent these damages, the logs may be treated with a biocidal product. 

As the treatment is temporary, use class is not relevant in this case. 

5.5.8.2.2.2 Temporary treatment of green timber 

This kind of treatment is used for the protection of freshly felled green lumber against 
colonization by blue stain and other discolouring micro-organisms (often named ‘sapstain’ as 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
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there are more than 200 fungi which can caused discoloration of the sapwood) and surface 
mould. 

A technical specification (CEN/TS 15082) is available. 

1  blue stain fungi and other discolouring sapwood fungi 

Blue stain is caused by microscopic fungi that only infect the sapwood. They can cause 
blue or grey discoloration of the sapwood, but have no impact on its strength. Blue stain 
reduces the value of the wood. 

Typical blue stain fungi are: Ceratocystis spp., Ophiostoma spp. Aureobasidium spp. 

Typical other discolouring fungi are: Stereum spp. 

In the final stage of processing in a sawmill, treatment with a biocidal product (commonly 
applied by dipping to prevent blue stain fungi) may be carried out. 

2  moulds growing often on the wood surface  

The major problems caused by moulds fungi are discoloration on surfaces, and 
sometimes health problems. They do not affect the strength properties of wood. 

Typical mould fungal genera on wood are: Alternaria, Aspergillus, Penicillium, 
Trichoderma. 

A dose rate / dipping time is part of the efficacy assessment. The label claim must 
mention the dose rate and the dipping time. 

5.5.8.2.2.3 Treatments of solid wood (EN 599-1 Standard) 

When the purpose is to protect the wood, a preventive treatment is often applied to prevent 
the degradation of wood by micro-organisms (for example fungi) and/or by insects (for 
example wood boring insects). The treatment type is related to the organisms against which 
the wood has to be protected and to the use class. EN 599-1 specifies what test should be done 
for each use class claimed.  

Different target organisms may preferentially attack either softwood or hardwood. Tests must 
be conducted on softwood and/or on hardwood as appropriate to the target organisms and 
following the requirements presented in the relevant test procedures.  

It must be noted that Use Class 1 requires only insecticide products and, starting from Use 
Class 2, products are fungicide alone or combine fungicide and insecticide activities.  

It may also be noted that in some cases when a claim against only blue stain fungi is made 
justified exemptions are possible 24.  

Use Class 1 

Required data 
Refer to EN 599 -1 table 1. 

Data  will  include  suitable  laboratory  data  using  treated  test  blocks  to  determine  the  toxic 
values against insects as appropriate. 

24 Products which only claim protection against blue stain can be authorized for uses where exemption of 
the requirement for efficacy against wood destroying fungi can be justified, e.g. for wood or wood 
products that by their nature are not susceptible to brown rot fungi. Pure anti-blue stain products may not 
be used together with product against wood destroying fungi to prevent double treatment of two 
fungicides. 

The test species used will depend upon the label claims and will include as a minimum the brown rot fungi 
and insects basidiomycetes and beetles spp. if appropriate (as in for Use Class 1. Use Class 1 products are 
only insecticides. 

Products used as wood preservatives with only insecticide activity can be authorised for preventive use 
only in UC1. For UC2 and higher classes, efficacy against brown rot fungi basidiomycetes must be 
demonstrated as a minimal requirement. This clarification (of interpretation of test species) should be 
considered to be effective immediately (and applying to on-going/past assessments) and not subject to 
the standard transitional period of 2 years for new guidance." 

 
 
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Data  should  be  presented  on  test  blocks  subjected  to  pre-conditioning  by  an  evaporative 
ageing process (e.g. EN 73). 

Test species  
The insect species tested will depend on whether a general or a specific efficacy claim is made. 
Data  should  demonstrate  activity  against  one  or  more  of  the  following  specific  insects  as 
indicator  species:  Hylotrupes  bajulus,  Anobium  punctatum,  Lyctus  brunneus,  and  where 
appropriate, termites. 

Note 

CAs  should  evaluate  the  available  data  to  determine  whether  they  are  sufficient  for  label 
claims as follows: 
a) for general claims against "wood boring beetles"25 
All  relevant  beetle  species  (Hylotrupes  bajulus,  Anobium  punctatum  and  Lyctus  brunneus) 
should be tested  except if data (relevant and robust literature data where the materials and 
methods  are  detailed;  certification  data26  on  a  case  by  case  basis)  are  provided  which 
demonstrate that one of the targets is the less sensitive or that the product has an equivalent 
activity against all beetle species (refer to EN599-1:2014, section 5.2.3) 

b) for claims against a specific beetle species 

If  claims  against  individual  beetle  species  are  detailed  on  a  product  label,  then  suitable 
efficacy data against those named target pests will be required. 

c) for claims against termites 

Some  data  on  efficacy  against  termites  will  only  be  required  when  the  product  is  to  be 
marketed for use as a termiticidal product or where local requirements demand such activity.  

For  a  product  claiming  activity  against  termites,  suitable  data  demonstrating  preventive 
efficacy against a European Reticulitermes species will be required. 

For a product claiming efficacy against overseas tropical termites, suitable data demonstrating 
preventive efficacy against relevant species will be required. 

Use Class 2 

Required data 
Refer to EN 599-1:2009 table 2. 

Data  will  include  suitable  laboratory  data  using  treated  test  blocks  to  determine  the  toxic 
values against the fungi and insects as appropriate. 

Test species 
The  test  species  used  will  depend  upon  the  label  claims  and  will  include  as  a  minimum  the 
brown rot fungi and insects if appropriate (as in Use Class 1). 

Note 

The  CAs  evaluate  the  available  data  to  determine  if  they  are  sufficient  for  label  claims  as 
follows: 

a) For claims against wood rotting fungi the following data have to be available: 

Suitable laboratory data demonstrating efficacy against brown rot fungi after ageing test in 
accordance with EN 73. 

b) For claims against wood discolouring fungi the following data have to be available: 

o  Suitable laboratory data on the protective efficacy  of the product against blue stain 
in  service  after  ageing  test  in  accordance  with  EN  73  or  after  a  natural  or  artificial 
weathering cycle as given in EN 152; 

o 

The  application  process  used  in  the  tests  (i.e.  whether  by  superficial  or  penetrative 

25  This  correction  has  been  made  for  an  error  in  drafting  and  should  be  considered  to  be  effective 
immediately and not subject to the standard transitional period of 2 years for new guidance. 

26 This certification ensures that products are fit for purpose and defines a capacity in the use of products 
taking into account among others the durability in the function (efficiency of the treatment). The efficacy 
part of the certification scheme is (in France) generated according the requirement of the EN 599. 

 
 
 
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treatment) has to be in accordance with label claims.  

c) For claims against insect pests the following data have to be available: 
As outlined in Use Class 1. 

Use Class 3  

Required data  
Refer to EN 599-1:2009 table 3a and table 3b. 

Data  will  include  suitable  laboratory  data  using  treated  test  blocks  to  determine  the  toxic 
values against the fungi and insects as appropriate.  

Test species 
The  test  species  used  will  depend  upon  the  label  claims  and  will  include  as  a  minimum  the 
brown rot fungi and insects if appropriate (as in Use Class 1). 

Note 

The  CAs  should  evaluate  the  available  data  to  determine  if  they  are  sufficient  for  claims 
matrix as follows: 

a) For claims against wood rotting fungi, the following data have to be available: 

o  Suitable laboratory tests as outlined for Use Class 2 and in addition, the efficacy will 
be  demonstrated  following  preconditioning  of  the  treated  test  blocks  by  a  suitable 
leaching procedure according to EN 84 

b) For claims against wood discolouring fungi the following data have to be available: 

o  Suitable laboratory data on the protective efficacy of the product against blue stain 
in service after a natural weathering or an artificial weathering as given in EN 152. 

o 

The application process used in the tests (i.e.  whether by superficial  or penetrative 
treatment) should be in accordance with label claims. 

c) For claims against insect pests (if relevant) the following data have to be available: 

As outlined in Use Class 1, and in addition the efficacy will be demonstrated following pre-
conditioning of the treated test blocks by a suitable leaching procedure according to EN 84 
if technically possible (i.e. this is not the case for EN 20-1 and 20-2 due to methodological 
constraints). 

According to EN599-1 field test results,  according to EN330 may be used by the applicant 
instead  of  certain  EN  113  test  results,  after  EN  84  leaching  test  to  derive  the  brown  rot 
fungi. They are not needed to derive the minimum retention requirements. 

Moreover EN 330 may be used as an alternative to basidiomycetes laboratory tests (EN 113 
+ EN 84) for product under coating. 

Use Class 4 

Required data 
Refer to EN 599-1:2009 table 4. 
Data  will  include  suitable  laboratory  data  using  treated  test  blocks  to  determine  the  toxic 
values against the fungi and insects as appropriate. In this situation available data should only 
include application of the preservative by penetrative treatments. 

Test species 
Test species used will depend upon the label claims and will likely include the following target 
organisms: brown and white rot fungi, soft rot micro-fungi and if relevant to label claims, blue 
stain fungi and insects as appropriate. 

Note 

The  CAs  should  evaluate  the  available  data  to  determine  if  they  are  sufficient  for  matrix 
claims as follows: 

a) For claims against wood rotting fungi, the following data have to be available 

o  Suitable laboratory data as outlined for Use Class 3 with the following supplements:  
-  all  laboratory  data  should  derive  from  impregnated  treated  test  blocks  (i.e.  a 
penetrative  treatment)  with  the  test  formulation  to  determine  the  toxic  values 

 
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against both brown and white rot fungi separately; 

-  a suitable laboratory test to determine the toxic efficacy against soft rot fungi and 

other soil inhabiting micro-organisms is required; 

b) For claims against wood discolouring fungi, the following data have to be available: 

o  A suitable laboratory test determining the protective efficacy of the product against 

blue stain for wood in service as given in EN 152. 

c) For claims against insect pests, the following data have to be available: 

o  As  outlined  for  Use  Class  1  and  in  addition,  efficacy  will  be  demonstrated  following 
pre-conditioning  of  the  treated  test  blocks  by  a  suitable  procedure  according  to  EN 
73 and to EN 84 separately). 

In Use Class 4 data (e.g. EN 252, literature, monitoring or other methods) will be provided 
to support the derived application rate.  

Use Class 5 

Required data 
Refer to EN 599-1 table 5. 

The principal agent of decay in this situation is the marine borers. Therefore in this Use Class 
available data must include evidence of efficacy in a relevant marine field trial carried out for a 
minimum of 5 years (e.g. to EN 275 or an equivalent test). 

The  decay  in  this  situation  by  basidiomycetes  fungi  does  occur  but  marine  soft  rot  fungi  are 
more  common  causing  surface  softening  of  timber.  Assessment  of  products  against  marine 
fungi  is  not  normally  conducted  using  routinely  laboratory  tests  because  of  the  difficulties  for 
providing  conditions  which  appropriately  model  the  marine  environment.  There  is,  at  present, 
not  a  recognised  standard  laboratory  test  for  assessment  of  timber  intended  for  use  in  salt 
water. 

Test species 
Test species used will depend upon the label claims. The principal agent of decay in the marine 
environment is the marine borers although claims against fungi can also be made. 

The CAs evaluate the data to determine if they are sufficient for label claims as follows: 

For claims against wood rotting fungi and marine borers, the following data have to be 
available: 

• For fungi available data as outlined in Use Class 4 as a surrogate has to be acceptable. 

• For marine borers, a relevant marine field trial data has to be carried out for a minimum of 5 
years according to EN 275 

5.5.8.2.2.4 Treatments of wood-based panels 

The biocidal treatment of wood-based panels is achieved either during or after the 
manufacturing process. 

During the manufacturing process, product can be included into the glue prior to application or 
directly by wood treatment. 

The evaluation of the durability of wood-based panels against brown rot fungi and white rot 
fungi should be carried out according to the ENV 12038 test method. 

There is no specific standardized methodology allowing the evaluation of the resistance of 
treated wood-based panels against soft rot or insects such as Lyctus spp. or termites. 
However, some of the existing standards usually applied to solid wood can be adapted to the 
evaluation of wood-based panels: CEN/TS 15083-2 (natural durability to soft rot fungi), EN 20-
2 (Lyctus spp.), EN 117 and EN 118 (termites). 

For post-manufacturing treatment, product can be applied by using a surface application 
process or pressure process. 

In that case, the EN 599-1 is appropriate for determining the retention of post manufacture 
treatment. 

 
 
 
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5.5.8.2.2.5 Barrier treatment against Serpula lacrymans 

The dry rot fungus (Serpula lacrymans = true dry rot fungus) occurs in buildings, causing 
brown rot in timber. The fungus can develop at relatively low wood moisture contents and is 
able to penetrate damp masonry over long distances in order to infect further timber or to 
develop its fruit-bodies. 

In general, in case of an infestation of Serpula lacrymans, the infected wood is cut away. To 
prevent the infection of the new placed wood with fungi coming from the surrounding masonry, 
a curative treatment against dry rot in walls (mortar) will result in creating a ‘preventive’ 
barrier in / on walls hindering the fungus to grow through. 

There is a specific Technical Specification (CEN/TS 12404) for determining the performance of 
a preservative applied to the upper surface of the mortar in preventing the growth of dry rot 
through the treated mortar when exposed to the fungus. This method is only applicable to 
masonry fungicides applied as a true solution of preservative. It is not applicable to rods, 
pastes and other similar preservative types. This method is applicable to preservatives applied 
to masonry by brushing, spraying and/or injection techniques or mixed into rendering and 
plastering mortar for masonry. 

5.5.8.2.2.6  Determination  of  preventive  product  application  rate  with  regard  to 
service life 

The evaluation of PT8 products efficacy is based on the retention of the product as determined 
in standard test methods, e.g. according to standards listed in EN 599-1. The values 
determined in this way are critical values (CV’s) for a particular formulation. The application 
rates derived from the CV’s are deemed to provide only a baseline efficacy and no conclusion 
on service life can be made. Indeed, neither is the term service life an absolute measure and 
no uniform mathematical model exists to derive such from CV’s, nor is determination / claim of 
a distinct service life part of the BPR. Estimation of service life (ESL) is based on the 
assumption, that different parameters have an impact on the service life of wood. This is 
explained in ISO 1586-1 and ISO 15686-2. 

An estimated service life of wooden products is influenced e.g. by local exposure conditions, 
maintenance, consumer expectation and long term experiences from field testing or industrial 
experiences. This can provide justification for setting higher or lower retention rates as derived 
from CV’s only. 

Because the concept of ESL is not part of the BPR and claims for a specific service life is 
consequently solely the applicant’s responsibility, the applicant must have the right to apply for 
lower or higher retentions than just the CV up to the retention rate which is limited by the 
human health and environmental risk assessments. 

In order to support his claim, for UC3 claims, the applicant should submit data from e.g. 
literature, EN 330. For UC4, the applicant will provide, EN 252 (applicable to UC4 claims) 
and/or other methods for justification. 

Particular specification for use class 4: 

The field tests sites (minimum two) or the data extracted from literature must be 
representative for climatic zones with regards to the markets targeted by the product. The 
selected sites must allow the evaluation of the product’s efficacy on all the biological organisms 
covered by the label claim. 

5.5.8.2.3 Curative treatment 

EN 14128 is the lead standard providing detailed insight into the minimum testing 
requirements for wood preservatives claiming curative activity. It must be noted, that testing 
standards concerning PT8 products are only available for testing against wood boring insects  

It is important to understand that conducting curative treatments may comprise 
series/combinations of different steps and application methods/techniques in order to achieve 
the desired result and quite often result in providing preventive and curative efficacy at the 
same time. 

 
 
 
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5.5.8.2.3.1 Wood boring insects 

Data  required  to  support  label  claims  for  curative  efficacy  may  include  some  tests  generated 
using existing EN standards for the relevant beetle species or other alternative supporting data.  

A number of EN standard tests exist for curative treatments for insecticides against Hylotrupes 
bajulus (ENV 1390) and Anobium punctatum (EN 48).The curative activity against Lyctus is not 
tested  separately  but  is  derived  from  results  from  testing  against  Anobium  punctatum  and 
Hylotrupes bajulus. 

5.5.8.2.3.2 Termites 

The control of termites enters into the scope of the PT8 and the PT18 depending of the use of 
the product. The definition of the product type is related to the use/mode of application of the 
product. 

The reader is also invited to refer to the PT18 efficacy (section 5.6.4). 

The curative treatments against termites are designed most of the time to kill the termite 
colony and prevent degradation of wood. 

We can distinguish treatment applied to wood, for example treatment of art furniture, wood 
rubble from treatment applied to other support than wood for example soil or masonry. 

If the product is applied on wood, then this product is covered by the requirement of the PT8. 
If the product is applied on another support than wood then it is covered by PT18. 

We can distinguish three groups of termites: 

•  Drywood termites (Cryptotermes, Kalotermes): Drywood termites live inside of the 

wood which is attacked. The curative treatments applied to the wood consequently 
destroy the entire colony. 

•  Subterranean termites (Reticulitermes, Coptotermes, Heterotermes): The core 

of the subterranean termite colony is located in the soil. Termite workers built tunnels to 
reach wood and destroy it. The treatment applied on infested wood kills the termites 
present inside of the wood but not the other members of the colony. 

•  Tree termites (Nasutitermes): Tree termites built epigeous (above-ground) nests, 

frequently on living trees. As a part of the colony has a subterranean location, termites 
infestations of wood in building may originate either from the nestmates located in the 
ground or in the epigeous nests. The treatment applied on infested wood kills the 
termites presents inside the wood but not the others members of the colony. 

5.5.8.2.3.3 Fungi 

Any claims for curative activity against wood rotting fungi will be supported by suitable efficacy 
data. No EN standard test protocols presently exist for curative treatments applied to wood. In 
general, as curative treatment, the infected wood is cut away. 

In all cases CAs evaluate the data available to determine if they are sufficient for supporting 
the label claims. 

5.5.8.2.4 Resistance 

Information on resistance and the likelihood of its development is required for BPR Annex I 
inclusion and is also demanded for product authorisation. 

At this point, no target organism resistance in field of chemical wood preservatives is known. 

More information on resistance can be found in Chapter 6.2 of this TNsG on Product Evaluation, 
in the Chapter 10 on the TNsG on the BPR Annex I inclusion and on the website of the 
Insecticide Resistance Action Committee and the Fungicide Resistance Action Committee 
(FRAC: http://www.frac.info). 

5.5.8.3 Biological re-testing after changing the product formulation 

While EN599-1 and EN 14128 provide the baseline for the testing requirements of new 
products, the corresponding annexes to both standards provide guidance on testing 
requirements when a formulation variation is caused by the addition, the substitution or 
removal of an active substance. Not all changes are subjected to re-testing and the informative 

 
 
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sections of the standards do allow the consideration and taking into account of other data on a 
case by case expert judgment basis without additional testing. These data sources are not 
defined in detail but could include: 

•  Literature data; 

•  Certification of the product by recognised national quality scheme systems e.g. 

CTBP+RAL; 

•  National registrations; 

•  Others. 

For any other changes in the formulation, refer to the informative annex A of EN599-1 and EN 
14128. An explanation of Annex A of EN599-1 can be found in Appendix 12. 

5.5.9 PT9 Fibre, rubber and polymerised materials preservatives 

The text for this section is under section 5.5.7 with PT7. 

5.5.10 PT10 Construction material preservatives 

Please  refer  to  the  General  sections  1-3  and  the  Preservatives  general  sections  (i.e.  5.5.1- 
5.5.3) of this guidance. 

5.5.11 PT11 Preservatives for liquid-cooling and processing systems 

Please  refer  to  the  General  sections  1-3  and  the  Preservatives  general  sections  (i.e.  5.5.1- 
5.5.3) of this guidance. 

5.5.12 PT12 Slimicides 

 Please  refer  to  the  General  sections  1-3  and  the  Preservatives  general  sections  (i.e.  5.5.1- 
5.5.3) of this guidance and the TNsG. 

5.5.13 PT13 Working or cutting fluid preservatives 

PT13 deals with preservatives for metal working fluids during their use in industrial processes. 
The general principles for evaluating PT13 products can be found in section 5.5.2 to 5.5.5. 
IBRG27 developed a method that allows to test the efficacy of active substances in a model 
matrix (“A Method for Determining the Basic Efficacy of Biocidal Active Substances used in 
Aqueous-Based Metal Working Fluids for their Protection in Use, IBRG3 FFG 16-001. This 
method should be used, unless it is justified that the method is not relevant for this specific 
product. 

27 International biodeterioration research group (IBRG): www.ibrg.org 

 
 
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5.6 Pest Control (Main group 3) 

5.6.1 General 

The text for this section is under development and will be added at a future update. 

Humaneness 
According  to  the  BPR  (Article  19(1)(b)  criterion  ii  and  common  principles  point  49  and  76  in 
Annex  VI)  biocidal  products  should  cause  no  unacceptable  effects  on  the  target  organisms, 
including  unnecessary  suffering  and  pain  for  vertebrates  (humaneness).  This  criterion  is 
relevant for biocides in the Pest Control PTs14, 15, 17, 19 (repelling or attracting vertebrates) 
and PT20. 

For these biocides an assessment must be made to demonstrate that the biocidal product does 
not  cause  unnecessary  suffering  in  its  effect  on  target  vertebrates.  This  must  include  an 
evaluation  of  the  mechanism  by  which  the  effect  is  obtained  and  the  observed  effects  on  the 
behaviour  and  health  of  the  target  vertebrates;  where  the  intended  effect  is  to  kill  the  target 
vertebrate, the time necessary to obtain the death of the target vertebrate and the conditions 
under which death occurs must be evaluated. 

A biocidal product intended to control vertebrates must not normally be regarded as satisfying 
criterion (ii) under point (b) of Article 19(1) unless: 

•  death is synchronous with the extinction of consciousness, or 

•  death occurs immediately, or 

•  vital functions are reduced gradually without signs of obvious suffering. 

For repellent products, the intended effect must be obtained without unnecessary suffering and 
pain for the target vertebrate. 

Guidance on the assessment of humaneness is currently not included in Volume II Efficacy Part 
B/C: Efficacy Assessment and Evaluation, but some general guidance can be found in the TNsG 
on Product Evaluation Chapter 6. 

5.6.2 PT14 Rodenticides 

General introduction 

This section provides guidance on the methodology for the evaluation of the efficacy of 
rodenticide biocidal products according to the common principles laid down in Annex VI of the 
BPR in order to demonstrate that the condition for granting an authorisation in Article 
19(1)(b)(1) of the BPR is fulfilled (i.e. the rodenticide is sufficiently effective). 

5.6.2.1 Introduction 

Depending on its intended purpose, a rodenticide may be regulated as a biocidal product or as 
a plant protection product28. This document covers the rodenticides under the BPR, which are 
used predominantly for the control of the house mouse (Mus musculus), brown rat (Rattus 
norvegicus) and the roof rat (Rattus rattus). Also other target species such as water voles 
(Arvicola amphibius), bank vole (Myodes glareolus), common voles (Microtus arvalis), field or 
wood mice (Apodemus spp.) and the grey squirrel (Sciurus carolinensis) are considered. 

The four standard fields of use are given below with examples of possible fields of use: 

28 Biocidal product (PT14): Rodenticides used for the control of mice, rats or other rodents (by means 
other than repulsion or attraction) outside plant growing areas, for example in farms, cities, industrial 
premises etc, and inside plant growing areas not to protect plant or plant products.  

Plant protection product: Rodenticides applied in plant growing areas (agricultural field, greenhouse, 
forest) to protect plants or plant products temporarily stored in the plant growing areas in the open 
without using storage facilities. 

Where a product is used in both situations (as PPP and BP), it will need dual authorisation for the relevant 
use in accordance with the last subparagraph of Article 2(2) of the BPR. See also 
http://ec.europa.eu/food/plant/protection/evaluation/borderline_en.htm 

 
 
 
 
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• 

in and around buildings 

o 

o 

o 

in and around residential homes and other places in which people are 
accommodated; 

in and around rooms intended for the preparation, processing or storage of food 
and beverages; 

in and around stores, ships’ holds, factories and silos; 

•  at waste dumps; 

• 

in sewers 
o 

in moist/wet environments such as sewers and watersides; 

•  open areas 

o  open areas such as airports or leisure areas. 
o  on animal husbandry farms (pigs, poultry, cattle, etc.); 

Since the majority of rodenticides are bait products, most of this guidance deals with the 
evaluation of the efficacy of baits. In the text it is indicated where it specifically concerns bait 
products or concerns other types of rodenticides. 

5.6.2.1.1 Aim 

The aim of this document is to provide guidance on how to assess the efficacy of rodenticides, 
in order to ensure that only sufficiently effective products are authorised and therefore placed 
on the market for use. Animal welfare considerations are also taken into account. 

5.6.2.1.2 Global structure of the assessment 

Full assessment of efficacy is conducted on applications for product authorisations. 

Information on effectiveness and intended use(s) of the product, together with its active 
substance(s), must be sufficient to permit an evaluation of the product and to define its 
conditions of use. 

Efficacy studies (see section 2 below for the type of testing required) should be performed with 
the product to evaluate whether the product is effective for the intended use(s) at the specified 
doses. Efficacy tests should be performed with the product (in its final formulation) for which 
the authorisation is sought, and the composition of the test-product should be provided in the 
efficacy reports (especially for field tests and palatability tests). Any efficacy data from 
scientific literature are considered only as supportive data and should not replace efficacy data 
obtained from efficacy tests, which should be performed according to recognised standards. 
Data on the mortality and, in case of bait products palatability of the bait, resulting from these 
studies are compared with the specified criteria. The basis for the evaluation is the uses 
specified in the application (i.e. draft SPC) submitted by the applicant. 

5.6.2.2 Dossier Requirements 

Data on efficacy are required for every application for authorisation. The following information 
on effectiveness is required for each biocidal product in accordance with Annex III of the BPR: 

1. Function (e.g. rodenticide) and mode of control (e.g. killing); 

2. Representative organism(s) to be controlled and products, organisms or objects to be 

protected; 

3. Effects on representative target organisms; 

4. Intended concentration at which the active substance will be used and application rate; 

5. Mode of action (including time delay); 

6. The intended uses for the product; 

7. Efficacy data to support these intended uses, including any available standard protocols, 
laboratory tests or field trials used including performance standards where appropriate 
and relevant; 

8. Any known limitations on efficacy: 

8.1. Information on the occurrence or possible occurrence of the development of 

resistance and appropriate management strategies; 

8.2. Observations on undesirable or unintended side effects for example, on beneficial 

and other non-target organisms. 

 
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Efficacy testing 

It should be noted that any efficacy testing conducted in the European Union on rodents should 
be in accordance with the principles set under Directive 2010/63/EU29 on the protection of 
animals used for scientific purposes. However, field trials with rodenticide products to control 
wild rodent infestations under actual use conditions that are carried out to demonstrate the 
results of already obtained data on palatability, mortality and humaneness are not considered 
animal procedures for the purposes of Directive 2010/63/EU. 

For all types of rodenticides, efficacy has to be demonstrated in a laboratory trial and a field 
trial or alternatively in a semi-field trial and a field trial for each target organism submitted in 
the application, unless specified otherwise in this guidance. For roof rats it is also acceptable to 
demonstrate efficacy: 

• 

in two or more well-conducted semi-field trials (for description see section 2.6 below), 
since in some regions infestations of roof rats are quite rare; or 

•  Two (or more) well-conducted field trial(s) in regions with infestations of roof rats. 

In general it applies that tests should be of high quality to be considered for evaluation. For 
animal welfare reasons, in laboratory tests, the number of animals per test should be 
restricted to a minimum. 

Positive results in field trials may outweigh negative results30 in laboratory studies, but only 
under the following conditions: 

•  there is at least one other laboratory study (or semi-field trial) with positive results for 

each study with negative results and; 

•  there is at least one field trial of high quality with positive results. 

Positive results in laboratory studies cannot outweigh negative results in field and semi-field 
trials. 

In case of testing only in semi-field or field trials (roof rats): 

•  at least two well-conducted semi-field tests or one field trial should have positive 

results, respectively. 

The following guidance is designed to be flexible and does not specify rigid protocols to which 
tests must be conducted. Published or unpublished data from any source will be considered 
provided the data are scientifically valid and relevant to the application. In all cases, the 
methods have to be described in sufficient detail to make the data reproducible. Ideally, data 
should be generated using national or internationally recognised testing methods and in 
accordance with the principles set under Directive 2010/63/EU on the protection of animals 
used for scientific purposes. However, applicants can also submit data generated using their 
own testing strategies where these are conducted and well reported to a sound scientific 
standard. In all cases, the data must allow a specific assessment of efficacy and, in case of bait 
products, palatability of the product. Anecdotal evidence will not be acceptable. 

Assessment will be made in relation to the effectiveness of the product for the intended uses in 
the draft SPC submitted with the application. This assessment will take into account the 
animals that are considered to be harmful and are to be controlled (target species), indoor or 
outdoor use, the method(s) of application, application rates, use patterns of the product, 
maximum storage period (shelf life) of the product, together with any other specific terms and 
conditions concerning the use of the product. 

The target species selected for efficacy testing should be appropriate to the geographic regions 
in which the product will be used. They should be named in the draft SPC for the product 
(either common or generic names may be used). Please note that in some countries specific 
rodent species are protected and no control action against them is permitted. 

Intended uses 

Examples of intended uses given in the draft SPC associated with the target organisms are : 

29  Directive  2010/63/EU  of  the  European  Parliament  and  of  the  Council  of  22  September  2010  on  the 
protection of animals used for scientific purposes. 

30 Negative results are those showing insufficient efficacy against the evaluation criteria (see section 4.1 
of this Guidance). 

 
 
 
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•  for use against house mice: 

o  this will require testing against Mus musculus. 

•  for use against rats 

o  this will require testing against Rattus norvegicus and Rattus. 

•  for use against brown rats 

o  this will require testing against Rattus norvegicus. 

•  for use against rats and house mice 

o  this will require testing against Rattus norvegicus, Rattus and Mus musculus. 

•  for use against rats in sewers 

o  this will require testing against Rattus norvegicus with specifically treated bait (see 

section 2.4 below) 

•  for use against voles 

o 

this will require testing against at least two vole species which differ in size and 
behaviour, for example, water voles (Arvicola amphibius), bank vole (Myodes 
glareolus) and common voles Microtus arvalis. 

•  for use against a field mice (wood mice) species 

o 

this will require testing against the specified target species, for example the 
long-tailed field mouse/wood mouse (Apodemus sylvaticus) or yellow-necked 
field mouse (Apodemus flavicollis). 

•  for use against [name of target species] 

o  this will require testing against the given target species. an example could be the 

grey squirrel (Sciurus carolinensis). 

General intended uses given in the draft SPC, such as 'for use as a rodenticide' or ‘for use 
against mice’, with no further clarification of the target species are not acceptable. This is 
because it would allow use against rodent species for which the product is not tested and/or 
not intended. Concerning the target species, intended uses have to be species-specific (both 
for products authorised for professional and non-professional users). 

Testing has to be species-specific, and for each target organism that is given in the draft SPC, 
a study should be conducted. This is because the biology, behaviour and susceptibility of target 
species, even within taxonomic groups such as rats, voles or mice, may differ considerably. For 
example, the brown rat (R. norvegicus) is more sensitive for anticoagulants than the roof rat 
(R. rattus), whereas it has been observed that the roof rat is more neophobic and will be less 
likely to accept baits than the brown rat. Mice are taxonomically very unspecific and may be 
applied to a broad range of species (e.g. Mus musculus, or various Apodemus species) with 
different biology, behaviour and susceptibility against the active substances. Vole species differ 
considerably in their size and habitat. Therefore, all target organisms given in the draft SPC 
have to be tested. If the authorisation of a rodenticide with a less specific intended use, such 
as ‘for use against voles’ or ‘for use against mice’ is applied for, the product has to be tested at 
least against all representative species of the respective taxonomic group. For voles there are 
products authorised under the plant protection products (PPP) legislation, but under some 
circumstances, there can be a need for biocidal product approvals (e.g. in case of invasions 
near buildings and disease spreading). 

Resistance claims are allowed for products based on actives with a mode of action other than 
anticoagulants. For products based on anticoagulants there is differing opinions of permitting 
claims by Member States31 and therefore, until further discussions and decisions are made, 
such intended resistance claims must be considered on a case by case basis in discussion with 
the Member States. An intended use such as ‘for use against rats and/or mice resistant to the 
first generation anticoagulants’, is generally not possible, because test animals which are 
resistant to first generation anticoagulants are difficult to define and their degree of 
susceptibility may vary. Moreover, when a case of resistance is recognised in a field situation, it 
is generally advisable to use non-chemical methods like mechanical or electronic traps, 
rodenticide with non-anticoagulant mode of action, or the most potent anticoagulant 

31  This issue is under review and discussion and  the  guidance  will be updated if the situation regarding 
resistance claims for anticoagulants changes.  

 
 
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rodenticides, and the use instructions in the draft SPC should generally contain a paragraph 
about resistance management. Therefore, a general intended use concerning resistance on an 
anticoagulant product may not be regarded as informative, since resistance generally refers to 
the active substance rather than a specific product. 

5.6.2.2.1  Test animals 

Although laboratory testing should preferably be performed on second generation wild animals 
housed in groups, the difficulty and constraints associated with obtaining and maintaining them 
for testing purposes is recognised. Therefore for tests conducted within the laboratory, animals 
sourced from recognised commercially available strains are acceptable. 

In accordance with Directive 2010/63/EU, Articles 7 and 9 and Section A, 3.2. of Annex III, , 
semi-field trials should preferably be conducted using wild rodents or their offspring. Although 
not preferred, it is possible to use strains that resemble wild strains in semi-field trials as an 
alternative. These strains should be outbred strains (e.g. Long Evans or Lister Hooded rats) 
which retain the behavioural characteristics of wild rodents, which includes neophobia, anxiety, 
and fully capable sensory organs (no impairment of seeing, hearing, smelling or taste). When 
laboratory strains that resemble wild strains are used, a short description of the behavioural 
characteristics as well as reasoning for the choice of the respective strain as test animals 
should be provided. Generally, the diet which rodents (laboratory and wild strain) receive prior 
to the tests can be crucial for their behaviour towards bait products. It is therefore important 
that, as far as possible, the study reports should also include information on the dietary history 
of the test animals. It is recommended that test animals should receive a rather broad diet 
during breeding. Where wild animals are used in laboratory or semi-field studies, these may be 
live trapped from the wild, reared in either outdoor colonies or under laboratory conditions 
such that it permits the animals to retain much of their natural physiological and behavioural 
characteristics. Breeding stock used for rearing wild rodents should not be selected for docile 
qualities or other characteristics that significantly alter their wild tendencies. 

OECD Guidance Document on the recognition, assessment and use of clinical signs as humane 
endpoints for experimental animals used in safety evaluation (OECD, 2002) must be 
considered. Unnecessary suffering must be avoided (e.g. excessive weight loss/severe 
dehydration, persistent convulsions, cannibalism/self-mutilation, etc.) and animals should be 
checked regularly. Moribund animals should be euthanized in line with the requirements to 
apply humane end-points by using clinical signs to determine impending death. 

Field trials should be conducted on wild rodent infestations and are not considered animal 
experiments provided the respective tests on efficacy, palatability and humaneness have been 
confirmed under controlled laboratory studies. 

The purpose of Article 62 of the BPR is to minimise the number of tests on animals and not 
duplicate any studies on vertebrates that might be required by the BPR. While the objective is 
clear for laboratory tests and semi-field trials, for which animals are used on purpose, for field 
trials the situation can be seen from a different perspective. Where a field trial is carried out 
under real life conditions and the rodents subject to such field trial would have been to be 
killed/controlled in any case by using other authorised products, then it is considered that such 
field trial does not involve any duplication of testing. Therefore, field trials for PT 14 would be 
exempted from Article 62 of the BPR. 

Concerning laboratory tests and semi-field trials, the objectives of Article 62 (of BPR) would be 
achieved by data waiving where there were already tests with a fully comparable bait 
containing an active substance with similar or lower toxicity (see Table 25 in section 5.6.2.2.7 
below). In such cases read-across could be accepted provided that, where relevant, a LoA 
(Letter of Access) is presented by the applicant. 

5.6.2.2.2  Laboratory studies for bait products 

For testing the efficacy of bait products, two types of laboratory studies are available, mortality 
tests (i.e. no-choice feeding tests) and choice feeding tests. Since mortality tests give very 
little information in addition to data from the bait choice feeding testing and in order to reduce 
the number of animal experiments, mortality tests (i.e. no-choice feeding tests) are not 
recommended and are not required. However, many applicants may have no-choice studies on 
their products as they have been conducted in the past. These can still be submitted as part of 
the data package but no new studies should be conducted. 

 
 
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Tests conducted to EPPO or the specimen protocol (Appendix 13 of this Guidance) are 
preferable but other data will be considered on their merits. The study must be representative 
for the treatment. Depending on the intended aim of the product, the house mouse, roof rat, 
brown rat or other species should be used as the test animal. Wild strain testing is preferable 
and is most important for the bait-choice test. However, since this is probably impractical for 
some applicants, an outbreed lab strain (e.g. CD rats) which is likely to exhibit traits of the wild 
strain is accepted as surrogate. 

Rodenticides with special indications, for instance foam products, which are taken up orally but 
are not bait products since they adhere to the rodent fur, require separate laboratory trials, 
where the conditions are properly simulated (see section 5.6.2.2.3 below). 

The bait choice feeding trials 

The aim of the bait choice feeding trials is to determine the palatability of the product for the 
test animal. If conducted on both fresh and aged product it may provide information on efficacy 
after a long period of storage of the product (see section 5.6.2.2.5 below). This test is 
preferably done with wild strain animals. In this test design, animals have the choice between a 
non-toxic food source (challenge diet) and the bait containing the active substance. Either the 
amount of bait consumed, in which the active substance is incorporated, or the mortality of the 
rodents is an indication that the bait is sufficiently palatable for a lethal dose to be ingested. 
Results are compared with the specified criterion (see section 5.6.2.4.1 below). 

Make sure that the challenge diet is a product that the rodent is accustomed to. 

Full details of the methods used should be provided and data should be presented to show the 
daily intake of both untreated diet and product, the palatability ratio (amount of product: 
amount of challenge diet) or product acceptance (amount of product eaten expressed as a 
percentage of total (product + challenge diet) consumption) for different sexes of rodent, any 
signs of poisoning and days to death, with appropriate statistical analysis. When no significant 
differences exist between the sexes, the data from the two sexes may be combined. Clinical 
observations should be conducted to determine mode of action, degree of suffering, duration of 
toxicosis prior to unconsciousness, etc. These data are optional but provide useful information, 
especially on new active substances. 

In some cases comparison with normal food intake is inappropriate. For instance when fast-
acting rodenticides cause a reduction in feeding activity or when only very small quantities of 
bait are required to cause effect. Therefore, the main criterion is not the percentage of 
consumed bait but the mortality resulting from poison uptake. 

Bait choice feeding trials with voles 

The test protocol for choice test against voles in the laboratory should be principally the same 
as for rats and house mice. 

5.6.2.2.3  Laboratory studies related to contact rodenticides and gassing agents 

Contact rodenticides 

The information that should be available in order to demonstrate efficacy will include: 

i)  Estimates of time to death from individually or group caged rodents exposed to the 

product for stated periods of time. Reference to EPPO Guidelines (EPPO, 1986) should 
be made.  

ii) Evidence from the laboratory that the target rodents will pick up the required dose from 

the application method is recommended. 

Gassing agents 

Rodenticidal gassing agents are typically used in gas-tight buildings, ships, airplanes, 
containers and storage locations or for burrow fumigation. The type of information that should 
be available in order to demonstrate efficacy will include estimates of the potency of the active 
substance and product by inhalation when applied as described in the use instructions in the 
draft SPC for the product. 

There are no internationally recognised standardised test protocols for testing efficacy of 
rodenticidal gassing agents. In general, the dossier requirements are the same as with bait 
products. No-choice tests are not necessary. The dossier should include simulated use-tests as 

 
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well as field tests. Simulated use tests should be conducted in gastight containers. The size of 
the container, duration of exposure as well as the concentration of the fumigant in the 
container should reflect a real-usage situation. 

It has to be noted that the use of gassing agents in sealed rooms, buildings, ships, airplanes or 
containers (generally denoted here as “rooms”) is different from use in burrows (generally 
denoted here as “rodent burrows”). Hence, it has to be declared for which use an authorisation 
is applied for. For each type of use a field study must be conducted. 

Generally, during each experiment the concentration of gas has to be monitored. The test 
reports should contain a detailed description of gas concentration, position of measurement 
points as well as the analytical method. The absence or presence of sorptive materials has to 
be documented. 

Field tests for burrow fumigants should follow the protocol for rodent baits. It has to be 
demonstrated that rodent populations in infected objects can be eliminated. The study has to 
include a description of the burrow (location in the infested object, position of entrance holes), 
for example, Ross, (1986), and Méthode CEB n°254 (2013) listed in Appendix 15 of this 
Guidance. The methods for a population census before and after application as well as the 
mortality criteria are the same as for bait products (see Appendix 14 of this Guidance). 

Field tests for rooms should include an estimation of the population size, but it is recognised 
that a feeding census is often not possible (e. g. in containers). In these cases, cages with the 
respective target organisms (mice, rats) should be introduced to the field object. Their 
placement should reflect the expected distribution of rodents in the object. It is important that 
some cages should be placed at spots which would represent “worst case scenarios”, i.e. places 
with air draft (since a room or container may not be perfectly airtight) or in hideouts. The test 
report should contain a detailed description of placement of the cages, as well as number, age 
and sex of the test rodents. Exposure time should be according to the use instructions in the 
draft SPC . After exposure, the number of dead rodents within the sealed room/compartment 
and/or inside the cages must be determined. Field tests with no scientifically comprehensible 
data on population reduction or mortality will not be accepted. In cases where a sufficient 
number of caged rodents have been introduced to field objects for efficacy testing, simulated 
use tests can be waived. The mortality criteria are the same as for baits.  

Considering the risks linked to the presence of rodents in an airplane, an efficacy of 100% is 
necessarily required. Indeed rats and mice (these latter being able to hide in places of low 
volume and completely inaccessible in airplanes) can cause damage, besides the problems of 
public health, which affect the safety of the airplane and the passengers. Besides possible 
damage linked to the urine on the electronics, these rodents possess incisors with continuous 
growth which oblige them to eat away permanently at any type of materials (threads, girdles, 
steering cables, printed circuits.). There is therefore no tolerance threshold, because a single 
rodent can cause irreversible damage. In order to make sure that the dose administered 
according to recommendations and within the framework of fumigation under actual conditions, 
achieves the required mortality concentrations, the following requirements have to be carried 
out: 

•  during fumigation, the measurements of the “CT” (measured effective concentration x 

time of fumigation) must be systematically taken. The aircraft to be fumigated may not 
be completely airtight and gas leaks may occur, therefore measures need to be taken 
for the required 100% efficiency; 

• 

• 

for every trial, the data for the calculation of the “CT” are to be collected from the start 
of fumigation with statements of concentration (two minimum test points according to 
the type of airplanes) made at regular intervals (frequency of five minutes) for the 
duration of fumigation as claimed by the applicant. It is suggested that these data 
should be collected for two operations of fumigation; 

to make sure that there is good distribution of the gas at lethal concentrations in the 
entire airplane, rats in individual cages (five rats per test point) must be placed next to 
all the concentration test points. This will allow estimation of the relation between the 
measurements, the “CT” and the mortality of the rodents; 

•  a statement of temperature and humidity should be made. 

 
 
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In case a gassing agent is used in combination with a specific device or is part of a device 
(e.g., traps), results from laboratory choice tests as well as (semi-) field tests should to be 
submitted. A no-choice test is not necessary; (semi-) field tests should have the same protocol 
as field tests for baits. A population census like in bait tests before and after application is 
needed. The mortality criteria are also the same as for baits. 

5.6.2.2.4  Laboratory studies related to specific efficacy claims regarding suitability 
of bait products for use in damp conditions 

Where it is claimed that a product is suitable for use in sewers or under damp conditions, the 
retention of palatability (such as the effect of the heat and humidity on palatability) should be 
tested in a choice test32 against all claimed target species, using product that has been 
specifically pre-treated to simulate such conditions. Please note that sewers are generally only 
infested by the brown rat. 

For this purpose, the bait product must be exposed to a warm and humid surrounding for at 
least five days. Bait which is pre-treated in such conditions, may be tested either with 
experimental animals or, preferably, in a semi-natural test system (pen test). The total number 
of animals should be 10 to 20. 

Below a preferred test protocol is described. Other test protocols will be considered on their 
merits and are acceptable provided they are scientifically justified. 

The bait portions/blocks must be weighed before treatment and then exposed to preferably 
30°C to 35 °C and 80 to 99% RH for five days. Stable conditions can best be achieved in a 
climate chamber. The bait should be placed in a water-permeable clay bowl, which itself is 
placed in a water-tight clay dish. The clay dish contains water, which permeates through the 
wall of the clay bowl with the bait, so that the surface of the clay bowl is permanently wet to 
simulate the moist surface of sewer walls. Each pre-treated bait portion/block is applied to the 
test animals for one day. The bait portions/blocks are then removed and replaced with new 
pre-treated bait. Since bait exposure to warm and humid conditions is for five days, the baits 
must be pre-treated stepwise, so that for each testing day, bait with exactly the same pre-
treatment time will be applied. The test chamber or test cage is not acclimatised, i.e. the test 
animals do not experience specifically warm or humid conditions. The bait is replaced daily with 
freshly pre-treated bait and is offered in a wet clay bowl to maintain surface moisture, so that 
the bait remains wet and does not dry out during the 24 h exposure to the test animals. 
Specific acclimatisation of test chambers/cages to high temperatures and humidity is therefore 
unnecessary and not advisable, as the test animals will most likely originate from laboratory 
colonies which are kept under normal conditions (i.e. moderate humidity and temperature). 
High temperatures and humidity may cause them to react with behavioural disturbances. 

To determine the bait consumption, bait is removed from the test chambers/cages each day 
and weighed back. After this, the bait should be dried, preferably by placement in a drying 
oven at 30 to 36 °C (note: since most bait blocks contain a significant portion of paraffin, the 
temperature for drying must not be too high). Bait portions/blocks are then weighed until no 
further weight decrease can be measured (i.e. the bait lost all water and is dry). 

To calculate the bait uptake, it must be taken into account that the initial weight of the bait is 
fresh weight, whereas the final weight after bait application to the rats and subsequent drying 
is the dry weight. Thus, the difference between both is not exactly the amount of bait 
consumed by the rats, since fresh baits may contain moisture (which adds to the fresh weight 
at the beginning of the experiment, but is removed after drying for the final weight 
determination). Hence, the water content of bait must be determined by placing five untreated 
bait portions for each product in a drying oven until no further weight decrease is determined. 
The difference between the fresh and dry weight is then taken into account for the 
determination of the amount of bait uptake (Equation [1]): 

Where: 

32  Field  tests  may  be  accepted  in  case  of  a  controlled  situation  without  re-entry  of  rats,  but  laboratory 
studies are preferred. 

 
 
 
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b is the amount of bait taken up 

f is the fresh weight of the bait prior to heat and humidity exposure 

d is the dry weight after bait application, consumption and drying 

w is the proportion of water content of the bait (determined through drying of untreated 
bait). 

The relative portion of bait taken up by the test animals in relation to overall food consumption 
can be then calculated as (Equation [2]): 

Where: 

c is the percentage of consumed bait during the test 

b the amount of bait taken up (corrected after Equation [1]) 

a is the amount of challenge diet taken up. 

5.6.2.2.5  Studies related to specific efficacy claims regarding to the shelf life of bait 
products 

When a bait product is claimed to be effective after a long period of storage, it is necessary to 
demonstrate that the product will still be effective and palatable after the stated storage period 
(i.e. shelf life). Analytical studies on active substance content are therefore not sufficient to 
support shelf life claims of bait products. 

Based on expert opinion, most bait products have been found to be effective and palatable for 
24 months (with preservatives) . Efficacy testing should therefore only be provided for: 

•  bait products with preservatives that claim a shelf life of longer than 24 months; 

•  bait products without preservatives that claim a shelf life of longer than 12 months; 

•  bait products for which the degradation of the active content is >10% and assessment 

of the degradation on the efficacy is needed to substantiate the shelf life claim  

For bait products with a shorter shelf life claim than stated above, no efficacy tests on aged 
bait (i.e. product at the end of maximum storage) have to be provided. For these products it is 
sufficient to provide tests on fresh bait (i.e. newly produced product). 

For bait products with a longer shelf life claim, the applicant must deliver data on the 
palatability of the product at the end of maximum storage for all target organisms claimed. The 
palatability of the aged product preferably is tested in bait choice feeding trials, but can be 
tested in field trials, provided these tests are scientifically valid (see section 2.6 below). 
Accelerated ageing studies, i.e. palatability studies in which the product tested is stored under 
challenging conditions, are not acceptable as these cannot simulate longer storage periods. 

5.6.2.2.6  Field trial and semi field trial 

The following text describes the field and semi-field testing of bait products, but is also largely 
valid for other rodenticide products. 

Field trials 

The aim of the field trial is to demonstrate the results on the effectiveness (palatability, 
mortality and humaneness) obtained during laboratory studies of the rodenticide product 
containing active substance under actual use conditions for the purposes of marketing 
authorisation. Field trials should only be performed once efficacy, palatability and humaneness 
have been confirmed in laboratory (semi-field) studies under Directive 2010/63/EU. 

Tests conducted to EPPO or the specimen protocols (Appendices 13 and 14) are preferable but 
other data will be considered on their merits. Depending on the intended use(s) of the product, 
populations of the respective target organisms (house mice, brown rats, roof rats or others) 
are used for this trial. 

Ideally, sites chosen for field trials should be representative of the range of locations where the 
rodenticide is to be used (indoor/outdoor), and should be infested with sufficient numbers of 
the target rodents so that the effectiveness of the product can be clearly demonstrated. It is 

 
 
 
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advantageous if the rodent infestations on the sites chosen are, as far as possible, discrete and 
not subject to potential rapid re-invasion. Rodent activity on the site should be determined 
before and after treatments using at least two standard techniques. 

Sketch maps of the sites approximately to an indicated scale showing all the important features 
including signs of infestation and location of rodenticide application should be provided. The 
amount of bait applied at each bait point and the distance range between bait points should 
correspond to those given in the draft SPC. Replenishment of the bait should follow intervals 
given in the draft SPC . Bait exposure should normally be for 4 days for acute products and 30-
40 days for multi-dose products after the first bait uptake or less when full control is achieved. 
Data should be presented to indicate levels of rodent activity both before and after treatment, 
amounts of bait consumed and all relevant information regarding treatment details. 

Semi-field trials 

As an alternative or addition to ‘field’ trials, evidence of the efficacy of a rodenticide product 
may be obtained with semi-field trials (otherwise referred to as pen trials). A semi-field trial 
simulates field conditions under controlled laboratory conditions. Bait acceptance and bait 
uptake in the field is strongly influenced by the social behaviour of the target species. Both rat 
species (R. norvegicus and R. rattus) as well as house mice (M. musculus) are social animals, 
and food exploration is largely social in these species. Hence, the most important field 
condition to be simulated is the presence of conspecifics, i.e. the semi-field trial has to be 
conducted with groups of rodents. Group size should be at least 10 animals in tests with both 
rat species and at least 10 animals in tests with house mice. Sex ratio should be approximately 
1:1 although single sex groups may be used with robust justification, e.g. to avoid 
unacceptable levels of aggression. Groups should consist of related animals to avoid 
intraspecific aggression. The test animals should either be directly caught in the field, or be 
bred from wild catches, as only wild-strain rodents show the typical behaviour of the target 
species which could be expected in the field. A test with laboratory strain rodents cannot be 
regarded as a proper simulation of field conditions. 

The test arena should provide shelter for the animals, as well as sufficient space for the 
animals to roam. The minimum space requirement would be ≥ 0.5 m² per rat and 0.25 m² per 
mouse. If possible, cage enrichment such as branches, ladders, tunnels and wooden nest boxes 
with nest material may be provided and details on this should be given in the test report. Cage 
enrichment should be designed in a way that daily inspection for dead rodents and spilled bait 
material and feed causes only minimum disturbance. 

The rodents have to be familiarised for at least three days with the test arena prior to bait 
exposure. The semi-field trial is always a choice test, and a suitable challenge diet must be 
provided together with the bait. The amount of bait applied should correspond to the amount 
given in the draft SPC. Bait exposure should normally be for 4 days for acute products and 30-
40 days for multi-dose products. Bait exposure must be followed by a 14 day post baiting 
observation period. 

Field tests with voles 

For efficacy testing of products against voles, the test protocols for house mice and rats are 
only suitable when the infestation is inside a building. Efficacy testing outside of buildings 
should be conducted with a specific protocol. In contrast to rats and house mice, voles 
excavate and inhabit galleries (tunnels beneath the surface) for food exploration and nesting. 

For each field test with voles, one test plot and one control plot should be investigated. 
Principally, the test protocol is the same for oral baits and gassing tablets/pellets. The pre-
treatment and post-treatment censuses are conducted by counting occupied galleries. For this, 
at least ten galleries should be opened on each plot (treatment and control). After 24 h, the 
number of refilled galleries is then counted. The number of refilled single openings is set into 
relation to the number of openings as an indicator for vole activity. Depending on the vole 
species, an alternative census method could be the closing of burrow openings. Reopening of 
burrows is then counted as a sign for activity. During the treatment, vole activity should be 
controlled after 5 and 10 days with the same method. 

Application of the rodenticidal product should follow the use instructions in the draft SPC. 
Normally, one bait portion has to be placed in each gallery. Replenishment of the bait should 

 
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follow intervals given in the use instructions in the draft SPC. Bait exposure should be for 14 
days. The efficacy is then calculated as (Equation [3]): 

Where: 

E is the efficacy, 

t are treated plots 

c are control plots,  

t1 and c1 are the ratios of refilled galleries/open galleries before treatment 

t2 and c2 are the ratios of refilled galleries/open galleries after treatment. 

Treatment and trials with oral bait should be undertaken in spring or autumn, as in the winter 
not much activity is to be expected, and in summer other food sources than the bait are too 
abundant. 

5.6.2.2.7  Waivers  

Waiving of laboratory trials or semi-field trials will reduce animal testing. For bait products, 
because the composition of the bait determines the palatability and hence efficacy of the 
product, even small changes in ingredients may affect the attractiveness. This may differ 
between target organisms and is difficult to predict in advance. 

Semi-field trials 

Laboratory testing of bait products (bait choice test or semi-field trial) should always be 
requested for new active substances, or if a product was altered regarding the active substance 
concentration and/or bait formulation. One exception would be if there were already test data 
with a fully comparable bait, i.e. containing a different active substance but otherwise the same 
or similar formulation with the same mode of action and similar or lower toxicity; (see Table 25 
below for a ranking of toxicity of existing active substances), in such cases read-across could 
be accepted; however if the two formulations contained the same active substance, then the 
concentration of the active substance would need to be the same. 

Field trials 

Field trials are always required when the composition of a product is changed. Exceptions could 
possibly include changes of minor importance in ingredients that are likely not to have an effect 
on palatability or efficacy, such as change in colour of a product. In case of waiving, the 
applicant needs to provide a robust justification why no testing was performed. 

Read-across between species is generally unacceptable unless the applicant can demonstrate 
that there is no significant difference in the susceptibility and behaviour of the species. 

Table 25: Toxicity ranking of known active substances used in anticoagulant 
rodenticides based on LD 50 (acute) data of brown rats and house mice compiled 
from CA-Reports, ranking from high (1) to lower toxicity (3)  

Rank of toxicity  Active substance 

1 

2 

3 

Flocoumafen, brodifacoum, difethialone 

Bromadiolone, difenacoum 

Chlorophacinone, warfarin, coumatetralyl 

5.6.2.2.8 Biocidal Product Families (BPF) 

A BPF of rodenticide baits may contain several bait products with different formulations, for 
example, various grain, block, paste and gel products. Each bait formulation should be 

 
 
 
 
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allocated to a different meta-SPC33. Each bait formulation within the BPF has to be tested, 
because it cannot be predicted which form is the least palatable. It would also be difficult to 
select one product that could be regarded as a ‘worst case scenario’ for testing all the 
formulations. Within a given meta-SPC, an individual product should only be tested to consider 
the minimum level of efficacy within the concentration ranges of the active substance in that 
meta-SPC. 

5.6.2.3 Methodology of assessment 

There are many standard test methods currently available that may be appropriate for the 
assessment of the effectiveness of rodenticides. A list of such test standards is presented in 
Appendix 15 of this Guidance. 

In addition to the standard test methods presented in Appendix 15, specimen protocols for a 
Choice Test and a Field Test are presented in Appendices 12 and 13 respectively. These 
Appendices are intended only to provide further information regarding the types of studies that 
may be utilised to assess the efficacy of some rodenticides, and some of the factors that should 
be taken into account. 

Any known limitations on efficacy (including resistance) should be considered during the 
assessment. Possible restrictions, risk mitigation measures, or recommendations concerning 
the use of the product in specific environmental or other conditions can be considered. Possible 
factors that can reduce the efficacy, for instance hot, cold or humid environments or the 
presence of other substances, in addition to the grounds for these should be stated. Possible 
recommendations concerning the avoidance of the continuous use of the product in order to 
prevent the selection and spread of resistant strains and the grounds for these (see TNsG on 
Product Evaluation and a report on risk mitigation measures for anticoagulant rodenticides as 
biocidal products 34) . State if the product cannot be mixed with, for example, other biocidal 
products or if the use of the product with other biocidal products is recommended. The 
guidance given on resistance for the corresponding data requirement of the active substance 
also applies here. The study results are compared directly with the criteria for efficacy (see 
section 4.1 below). 

5.6.2.4 Assessment of authorisation 

5.6.2.4.1 Norms and criteria 

In accordance with Article 19(1)(b)(1) of the BPR, a biocidal product may only be authorised if 
it is sufficiently effective. This is implemented in the following way. 

In general rodenticide products are normally considered to be sufficiently effective if the 
following results can be achieved: 

• 

• 

required results in laboratory test and semi-field test: 
o 

90% mortality within a relevant time frame 

required results in field test: 
o  Monitoring of the test population should show a 90% decrease of the population 

Rodenticide bait products are considered to be sufficiently effective if the following results can 
be achieved: 

• 

required results in the bait choice feeding test, semi-field test and sewer test (if 
claimed): 
o 

90% mortality. The percentage of ingested bait containing the product should be 
normally 20%, but it may be lower because a mortality of 90% the product would 
still be effective. In case of a bait ingestion <20%, justification should be provided. 

•  required results in field test: 

• 

feeding on census bait after treatment should be reduced by at least 90% from the 

33 See Q&A pair number 6 in Annex IV of the Note for guidance "Implementing the new concept of 
biocidal product families" (CA-Nov14-Doc.5.8 – Final.rev2). 
[https://circabc.europa.eu/w/browse/c309ae58-bdd7-421d-a678-8d8ac361d4e0]  

34  “Risk mitigation measures for anticoagulant rodenticides as biocidal products” 
[https://circabc.europa.eu/sd/a/343a61cd-b8d4-40af-9e5c-4f763aea3240/CA-Nov14-Doc.5.1%20-
%20draft_final_report_RMM.docx6]. 

 
 
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levels of feeding on census baits before treatment. When other types of quantitative 
monitoring of the test population are used, such as tracking activity measurement 
and census by trapping, they should sufficiently show the decrease of the 
population (90%). 

The efficacy of the product after a specified storage time (e.g. shelf life as claimed in the use 
instructions in the draft SPC) is also taken into account when assessing efficacy of a rodenticide 
bait. 

Deviations from the norms are possible, but must be justified in the application. The 
Competent Authority will evaluate any justification on a case-by-case basis, consulting the 
other CAs where appropriate and decide whether it is acceptable or not. 

In order to promote the development of new types of products (less toxic, more humane), a 
mortality <90% could be acceptable when the product is used as an accompanying method, 
(i.e. used with another product to demonstrate efficacy), but not as a stand-alone product. 
However, mortality of these new type of products should not be <50%. The use of a product as 
an accompanying method should be reflected in the use instructions in the draft SPC. 

For the assessment of resistance, reference is made to TNsG on Product Evaluation . 
Information on resistance testing techniques is also available from the Rodenticide Resistance 
Action Committee (RRAC)] and Prescott et al. (2007). 

5.6.2.5 References for PT14 

Prescott, C. V., Buckle, A. P., Hussain, I. and Endepols, S. (2007) A standardised BCR 
resistance test for all anticoagulant rodenticides. International Journal of Pest Management, 53 
(4). pp. 265-272. ISSN 0967-0874.  

Rodenticide Resistance Action Committee, RRAC. A Reappraisal of Blood Clotting Response 
Tests for Anticoagulant Resistance and a proposal for a standardised BCR Test Methodology 
[www.rrac.info]. 

Ross, 1986. Comparison of fumigant gases used for rabbit control in Great Britain. Proceedings 
of the Twelfth Vertebrate Pest Conference (1986). 
[http://digitalcommons.unl.edu/cgi/viewcontent.cgi? article=1053&context=vpc12]. 

5.6.3 PT15 Avicides, PT16 Molluscicides, vermicides and products to control 
other invertebrates & PT17 Piscicides 

Please refer to the General sections 1-3 of this guidance and the TNsG.  

For product-type 16, EPPO guidelines for efficacy testing are highly recommended (e.g. EPPO 
guidelines 95 for molluscicides in terrestrial environment). 

5.6.4 PT18 Insecticides, acaricides and products to control other arthropods 

5.6.4.1 Introduction 

Depending  on  its  field  of  use  a  product  to  control  insects  and  other  arthropods  may  be 
classified as a biocidal product or plant protection product. This section covers the products to 
control insects and other arthropods in the category of biocides, which are products against all 
pest arthropods except those that are plant parasitic. 

This first section gives a general introduction. The following sections describe per insect or per 
type of use what the  requirements for  efficacy  testing are. Information is missing on  some  of 
the  organisms  to  be  controlled  with  these  products  and  also  some  of  the  uses  and  types  of 
products. For instance, little information is provided on treated articles (e.g. insecticide treated 
mosquito nets etc.). These data gaps will be filled in a future update of this guidance. 

5.6.4.1.1 Aim 

The  aim  is  to  assess  the  efficacy  of  biocidal  products,  to  ensure  that  only  effective  products 
enter the market. 

 
 
 
 
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5.6.4.1.2 Global structure of the assessment 

A full assessment of efficacy is conducted for applications for product authorisations.  

Factors,  which  are  taken  into  consideration  during  assessment  of  the  efficacy  for  a  biocidal 
product to control insects and other arthropods for which authorisation is sought, are: 

• 

• 

• 

• 

• 

• 

• 

the target organism to be controlled; 

the physical state in which the product is applied (e.g. liquid/powder/bait); 

the areas of use, these may be: 

in and around residential homes and other spaces in which people are accommodated; 

in and around spaces in which animals are accommodated 

in spaces intended for the preparation, processing or storage of food and beverages; 

in empty stores, ship’s holds, factories and silos. 

Information  on  effectiveness  and  intended  uses  of  the  product,  together  with  its  active 
substances, must be sufficient to permit an evaluation of the product, including the nature and 
benefits that accrue following use  of the product in comparison to  suitable reference products 
or damage thresholds, and to define its conditions of use. 

A combination of laboratory studies, rigorous simulated-use laboratory studies, or field studies 
can  be  used  to  evaluate  whether  the  product  is  effective  for  the  requested  use(s)  at  the 
specified doses. Data from these studies are compared with the specified criteria.  

Assessment  will  be  made  mainly  in  relation  to  the  claims  for  the  effectiveness  of  the  product 
made on the product label. This assessment will take into account the pest(s) to be controlled, 
indoor  or  outdoor use,  the method(s)  of application, application rates  and use patterns of the 
product, maximum storage period of the product, together with any other specific claims made 
for  the  product.  More  information  on  different  aspects  of  the  label  claim  can  be  found  in 
Appendix 1. Appendix 17 shows examples of possible label claims. 

5.6.4.1.3 Dossier requirements  

Data on efficacy are required for every application for authorisation.  

The following guidance is designed to be flexible and does not specify rigid protocols to which 
tests  must  be  conducted.  Published  or  unpublished  data  from  any  source  will  be  considered 
provided  the  data  are  valid  and  relevant  to  the  application.  In  all  cases,  the  methods  and 
results have to be described in sufficient detail to make the data reproducible and to allow a full 
assessment. Anecdotal evidence will not be acceptable. 

Ideally, data should be generated using internationally recognised testing methods (ISO, CEN, 
OECD,  WHO  etc.).  Several 
for 
international  standard  test  methods  currently  exist 
insecticide/acaricide products. A list of these is presented in Appendix 18 to this document. 

If there are no guidelines available or guidelines  are not suitable, the applicant may use their 
own methods (intra-company Standard Operating Procedures), on condition however, that the 
studies  are  scientifically  robust,  well  reported  and  provide  a  clear  answer  to  the  question.  In 
addition, the test methods applied and the test conditions should be clearly and fully described 
and  must  address  the  efficacy  claim  that  appears  on  the  product  label.  The  use  of  existing 
guidelines,  with  revisions  to  make  the  guideline  more  suitable  for  the  specific  product  or 
company conditions, is also possible. 

For each test information such as the following should be available: 

• 

the  names  of  actives  substances  and  their  respective  concentration  in  the  tested 
formulation;  

•  as the formulation may be very important for  the efficacy, if the test  item differs from 

the product to be authorised, its composition should be provided; 

•  a statement about what is expected from the test, what should be determined and with 

which precision. Power and sample size considerations should be included as well; 

•  description  of  the  test  conditions  (size  of  cage,  floor  area,  presence  of  harbourages, 
presence  of  (alternative)  food,  water,  temperature,  photoperiod,  location,  weather 
conditions); 

 
 
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•  are the test organisms allowed to acclimatise to the test conditions before the test? For 

how long? 

•  how many test organisms are present (sample size)?  

•  describe population composition (males, gravid or non-gravid females, nymphs, larvae, 
age  of  the  population  or  generation  number  F1,  fed  or  unfed)  noting  that  the  feeding 
behaviour of some insects (i.e. Blattella) changes during their life; 

•  are the test organisms starved prior to the test? 

•  are  field  strains  or  known  insecticide-resistant  strains  tested  (claim  “effective  against 

strains resistant to x”)? 

•  a description of the history and origin of the test strain; 

• 

is bait consumption determined? If so, a covered bait should be included to determine 
weight  loss  due  to  evaporation  to  correct  weight  loss  of  the  exposed  bait  for  actual 
consumption; 

•  are one or more alternative baits (e.g. registered reference products) or alternative food 

source present in the same test container or protocol? 

• 

• 

• 

raw data should be available for each study, rather than just a summary of the results; 

show  the  results  of  both  tests  (with  biocide)  and  control  (without  biocide)  treatment, 
preferably in a table; 

size of the test population in the field before and after the test; 

•  description of the monitoring methods used before, during and after the test; 

• 

statistical methods, if appropriate. 

5.6.4.1.3.1 Test design 

Although in general nationally or internationally recognised testing methods are preferred it is 
not always possible to use these. For some products no standard methods are suitable. In that 
case a test has to be designed. 

Various factors must be considered when designing the tests, for example the number of test 
individuals  (insects,  mites,  other  arthropods)  needed.  The  ultimate  aim  of  relevant 
considerations should be to design experiments that economise on test individuals, but on the 
other hand generate sufficient power to detect effects of a magnitude considered important to 
demonstrate.  To  save  test  individuals,  replicate  tests  are  conducted.  Another  argument  for 
using  replicates  is  to  account  for  the  variation  among  test  individuals  in  susceptibility  and 
responses  to  the  biocides.  Numbers  of  test  individuals  per  replicate  group  and  dose  level 
(treatment  group)  as  well  as  the  number  of  replicates  in  the  entire  study  need  to  be 
established prior to conducting the tests. As the improvement in power wears off substantially 
as the number of replicates increases beyond five, it is usually sufficient to conduct four or five 
replicate tests at each dose level, employing 10 (or 20) test individuals each. The precise needs 
will  depend  on  the  size  of  the  variances,  relative  and  absolute,  between  and  within  the 
replicates.  This  can  differ  between  insect  species  and  test  design.  Sample  size  should  be 
adequate to detect differences among groups (untreated vs treated) with a statistical power of 
at least 80%. Some details on these issues are outlined at the end of each section. 

Useful information on the principles of test design, analyses end evaluation of efficacy trials can 
be found in the EPPO standards pp1/152(3) and pp1/181(3). 

5.6.4.1.3.2 Test examples 

In the following sections (5.6.4.1.3.2 to .15) examples are given of what kind of tests can be 
expected for efficacy testing. Sometimes these examples are a summary of a standard test, in 
other  cases  a  company  test  is  described  or  a  general  idea  of  what  the  test  should  be  like  is 
given. There is a great variation in how specific the description is. For instance, the number of 
replicates is given only when this was determined in the test described. 

In  all  cases  these  tests  are  only  meant  as  examples,  not  obligatory  requirements.  Since 
products  against  insects  and  other  arthropods  are  so  diverse  in  application  method,  mode  of 
action etc. the guidance cannot possibly cover all possible ways of controlling arthropods. 

 
 
 
 
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5.6.4.1.3.3 Laboratory versus (semi) field trials 

Laboratory and field trials with the test arthropods are normally needed to assess the efficacy 
of  the  product.  Field  trials  are  not  mandatory  in  some  cases,  as  outlined  in  the  sections  on 
specific  groups  of  arthropods  below.  In  some  cases  when  robust  field  studies  are  available, 
laboratory studies can be waived. If the product is applied as a bait, the entire bait, including 
the bait-box if applicable, should be tested, not only the product which is contained in the bait. 
When efficacy against several insects or other arthropods is claimed not all organisms have to 
be tested when appropriate bridging studies are available. 

In the case of field trials where true replication is almost certainly impossible to achieve, a full 
description  of any factors that might be expected to influence product performance  should be 
given.  These  may  include  the  risk  of  re-invasion  from  adjacent  areas,  general  levels  of 
sanitation, treatment history etc. and are intended to provide the authorities with information 
to assist with the interpretation of the results obtained. 

In the following sections (5.6.4.1.3.2 to .12) more specific dossier requirements are given per 
pest  species.  In  most  cases  a  general  description  of  a  proposed  method  is  provided.  This  is 
only  to  give  an  idea  of  what  kind  of  tests  should  be  provided.  More  detailed  descriptions  of 
tests can be found in the standard test methods (norms) listed in Appendix 18. This is a list of 
all available methods (as far as we know now) without distinction on usefulness, repeatability, 
order  of  acceptability  or  robustness.  Some  norms  might  have  a  different  approach  than 
described in the section for that insect. If this approach is more suitable for the product under 
investigation the norm should be used. 

5.6.4.1.3.4 The importance of controls on efficacy studies 

The importance of control experiments for efficacy studies must be stressed with regard to the 
efficacy evaluation. Studies should be conducted alongside negative controls wherever possible 
to provide a reference point for the treatment results. A useful definition of this term is given: 
“A  negative  control  situation  may  be  one  in  which  the  experimental  design  of  the  study  is 
identical to that of the biocide challenge test except that the biocidal agent is not applied in the 
control study. A biocidal agent may be considered as the formulation or as the actual biocidal 
active ingredient itself.” 

The negative control trial should normally be of similar size (i.e. number of replications) as the 
test  itself,  to  make  statistical  comparison  possible  and  to  get  a  fair  impression  of  control 
mortality.  

A relevant reference product (authorised, commercially available) can often be included at label 
rates in a protocol for laboratory and/or  field studies as positive  control. Unfortunately at this 
moment  no  standard  reference  products  are  available,  however,  an  authorised  reference  can 
be included. 

It  is  recognised  that  generation  of  such  control  data  can  be  relatively  straightforward  in  well-
defined  test  situations  such  as  laboratory  and  simulated-use  tests.  However,  it  is  also 
recognised that this can present a problem in field situations, where  control  sites may not be 
environmentally equivalent to the treatment site. 

In such instances, there may be an alternative means of generating reference data other than 
collecting  data  from  an  untreated  site.  This  method  may  involve  pre-treatment  monitoring  of 
the  site  in  question.  This  monitoring  must  be  quantitative,  e.g.,  assessment  of  numbers  of 
trapped insects. In these instances, a  ‘baseline’ infestation level would be established through 
such  monitoring  and  then  the  effect  of  treatment  on  this  baseline  can  be  assessed.  Post-
treatment monitoring is required for this method. 

5.6.4.1.3.5 Specific data to support label claims 

In  assessing  the  efficacy  of  a  biocidal  product  to  control  insects  and  other  arthropods 
competent authorities should in particular take the following parameters into account: 

• 

target organisms/spectrum of activity; 

•  mode of action/effect;  

•  use patterns/methods of application; 

•  dose rate. 

 
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The  data  provided  in  support  of  the  efficacy  claims  must  be  sufficient  to  cover  these  key 
parameters. 

5.6.4.1.3.6 Examples of specific label claims with respect to target organisms 

For  specific  target  pests  where  only  efficacy  against  one  insect/arachnid  order  or  a  certain 
family  within  that  order  is  claimed,  data  against  only  a  limited  number  of  pest  species  will 
normally be required. To illustrate this point, a number of examples are given below: 

•  FOR USE AGAINST FLEAS - Data against the cat flea (Ctenocephalides felis) or the dog 

flea (C. canis) should normally be available; 

•  FOR  USE  AGAINST  COCKROACHES  -  Data  against  two  key  species  such  as  German 
cockroach  (Blattella  germanica)  and  the  oriental  cockroach  (Blatta  orientalis)  should 
normally be available; 

•  FOR USE AGAINST DUST MITES  - Data against  Dermatophagoides sp. should normally 

be available. 

In  the  European  tropical  overseas  regions,  the  most  common  genus  encountered  could  be 
different.  A  specific  claim  should  therefore  be  proposed,  with  referred  target  organisms.  This 
special request could concern for examples termites, cockroaches or mosquitoes. 

5.6.4.1.3.7 Examples of broad label claims with respect to target organisms 

Broad  label  claims,  such  as  "crawling  insect  killer"  or  "flying  insect  killer",  should  be 
accompanied  by  qualification  of  the  range  of  pests  against  which  the  product  may  be  used. 
When broad claims are made, data on representative pest species will need to be provided for 
the range of pest orders against which efficacy is claimed. 

Representative  pests  from  these  orders  will  have  to  be  appropriate  to  the  use  pattern  of  the 
biocidal product i.e. the environment of the areas to which the biocide is to be applied and the 
nature  of  the  application  (e.g.  whether  it  is  a  space  application  or  surface  application)  will 
define the most appropriate pests to be tested. 

For  each  order  stated,  at  least  the  principal  target  species  will  need  to  be  tested  for  public 
hygiene use, before a  general  claim is likely to be supported. In more  specific areas, such as 
use  against  stored  product  pests,  data  on  at  least  two  major  representatives  of  the  orders  in 
question will normally be needed before a general claim is likely to be supported. 

Where such a claim covers a diverse range of pest habitats and pest morphology and biology, 
data  from  a  greater  number  of  representative  species  will  need  to  be  provided.  Appendix  17 
shows examples of possible label claims and the test species required. 

When  cockroaches  are  used  as  a  reference  species,  it  can  only  be  used  for  the  general  claim 
“crawling  insects”.  If  efficacy  against  other  insects  are  claimed  specifically  (e.g.  crawling 
insects including bed bugs) tests against these  other insect  should also be provided. Also if  a 
company wants authorisation for more specific use with the same product they have to present 
specific  data  on  the  specific  pest  they  are  claiming.  This  is  a  consequence  of  the  use  of 
“reference species”, which should not be a way of short-circuiting the evaluation for efficacy. 

5.6.4.1.3.8 The distinction between professional and consumer products 

In  some  cases  the  dossier  requirements  and  norms  and  criteria  for  the  evaluation  may  differ 
between professional and consumer products. Products used by professionals must have a high 
level  of  efficacy  since  the  objective  is  to  eradicate  the  infestation.  For  consumer  products  an 
immediate  knockdown  is  often  more  important  than  eradication,  of  course  depending  on  the 
claim.  For  instance  a  spray  against  cockroaches  does  not  necessarily  have  to  eradicate  the 
whole  population  but  it  should  work  fast.  Consumers  want  to  see  that  the  insect/arthropod 
dies/knocks  down  immediately  after  they  spray.  For  consumers  it  is  difficult  to  eradicate  a 
whole  cockroach  population  since  reinvasion  from  other  premises  will  take  place,  therefore 
eradication  does  not  always  have  to  be  proven.  For  each  pest  group  it  will  be  listed  whether 
requirements differ for consumer and professional products. 

5.6.4.1.3.9  The  distinction  between  principal  target  and  secondary/incidental  target 
pests 

 
 
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Screening  tests  (see  sections  below  for  details)  can  be  used  as  bridging  studies,  showing 
similar  effect  of  the  product  to  different  pest  species,  after  which  in  some  cases  field  studies 
can be waived for secondary target pest species. 

5.6.4.1.3.10 Claims for residual efficacy 

Most  insect/arthropod  pests  are  cryptic  and/or  nocturnal  in  behaviour  and  are  unlikely  to  be 
contacted  directly  by  a  spray  during  application.  For  this  reason  many  control  programmes 
involve the use of relatively stable active substances applied to buildings and other surfaces to 
leave  residual  deposits.  These  compounds  are  intended  to  remain  chemically  active  and 
therefore  effective  for  periods  of  weeks  up  to  several  months  following  treatment,  i.e.  they 
have a high residuality. Residual life is a term to describe the period during which the biocide 
will  be  present  in  sufficient  quantity  to  kill  target  pests,  which  walk  upon  it  for  a  sufficient 
period of time to pick up a lethal dose. 

Thus the amount of biocide residue deposited on treated surfaces is critical to the effectiveness 
of  many  treatments  against  crawling  (and  flying)  pests.  Ideally,  the  amount  of  residue 
deposited  should  be  determined  for  instance  by  calculation  or  under  actual  or  simulated  use 
conditions. The method(s) of determination must be available with the test data. 

Residual efficacy must be proven in tests. Usually, laboratory testing is performed to establish 
the efficacy direct after application and at the end of the residual life of the product. 

The  types  of  surfaces  to  which  residual  products  are  applied  must  be  reported  since  surface 
type has a pronounced effect on the amount of active residue available to pests. In general a 
selection of both absorptive and non-absorptive surfaces, related to the label claim, should be 
tested when supporting a residuality claim for crawling (and flying) pests. These could include 
vinyl  tile  or  linoleum,  stainless  steel,  painted  and  unpainted  wood,  carpet,  concrete  and 
ceramic tile. 

Efficacy  data  submitted  to  the  competent  authority  in  support  for  residual  treatments  should 
indicate the appropriate dosage and the utility of the formulation when used as directed. 

5.6.4.1.3.11 Residual treatments may also involve the use of palatable baits. 

When a bait product is claimed to be effective after a long period of storage, it is necessary to 
demonstrate  that  the  product  will  still  be  effective  and  attractive  after  the  stated  storage 
period.  The  applicant  must  either  submit  data  for  palatability  of  the  product  at  the  end  of 
maximum  storage  or  alternatively  (in  case  of  a  new  product)  data  for  a  stress  test  with 
'accelerated ageing', i.e.  a palatability test with the product which is  stored under challenging 
conditions (see FAO accelerated test). 

5.6.4.1.3.12 Claims relating to outdoor use 

When products are intended for outdoor use, tests should normally demonstrate efficacy under 
outdoor conditions. Changes in temperature and rainfall can have effect on the efficacy of the 
products. In general field trials cover this outdoor use. In some cases a field trial can be waived 
when a laboratory test can be done under worst case conditions. 

5.6.4.1.3.13 Mode of action 

There are a variety  of  modes  of action and possible  effects  on target  organisms derived from 
the  proposed  use  of  a  product  to  control  insects  and  other  arthropods.  The  available  data 
should give brief details to indicate the route and nature of the action (e.g. whether action is by 
contact  or  stomach  poison),  and  the  nature  of  the  effect  (e.g.  cholinesterase  inhibitor,  chitin 
synthesis  inhibition,  juvenile  hormone  analogue  giving  rise  to  sexually  immature  adults  or 
supernumerary nymphs). 

A  variety  of  molecules  exist  which  control  invertebrate  pests  by  preventing  successful 
completion of the insect's life cycle, rather than being acutely toxic to the insect. Examples of 
such molecules include chitin synthesis inhibitors (CSI) and juvenile hormone analogues (JHa). 
The  CSI  act  by  disrupting  the  deposition  of  chitin  during  the  formation  of  the  insect’s  larval 
cuticle  after  moult,  whereas  JHa  aim  to  interfere  with  the  hormone  based  control  of 
metamorphosis and reproduction. These two types of molecules are often referred to as insect 
growth  regulators  (IGR)  to  distinguish  them  from  conventional  insecticides  with  neurotoxic 
action. 

 
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Consequently molecules that affect the developmental cycle of insects may be effective without 
resulting in the immediate death of the insect and therefore efficacy trials should be designed 
to  address  the  most  appropriate  life  cycle  stage  of  the  insect  sensitive  to  the  molecule  of 
interest  and  also  to  measure  any  long  term  effects  (e.g.  on  the  fertility  and  fecundity  of 
females or any effects on the embryonic development in the egg stage). 

For  example,  in  measuring  the  effectiveness  of  JHa,  trials  should  be  designed  to  record  the 
number  of  adults  produced  from  treated  nymphs/larvae,  the  number  of  adults  with  deformed 
wings or terminalia and the mortality of insects prior to and at metamorphosis. Additionally a 
number  of  newly  moulted  females  should  be  selected  randomly  from  each  treatment 
dose/formulation and their ability to produce viable eggs/oothecae after pairing with untreated 
males should be recorded. 

IRAC,  the  Insecticide  Resistance  Action  Committee,  has  developed  a  classification  of 
insecticides based on mode of action (www.irac-online.org ). 

5.6.4.1.3.14 Resistance 

Information  on  resistance  and  the  likelihood  of  its  development  is  required  for  BPR Annex  I 
inclusion and is also important for product authorisation. 

For  insecticides  resistance  can  be  a  problem.  Some  pests  are  more  capable  of  building  up 
resistance  then  others.  For  instance  flies,  with  multiple  generations  and  multiple  females  that 
can lay many  eggs,  resistance  can be  expected to build up easily. In  ants on the  other hand, 
with one or few queens who lay eggs for a long period, and a biocide that kills the whole colony 
most of the time, it is not to be expected that resistance will build up. Therefore, a resistance 
management  strategy  has  to  be  provided  for  flies  but  not  for  ants  for  evaluation  at  product 
authorisation. 

A  resistance  management  strategy  is  generally  based  on  the  use  of  two  modifiers,  the 
frequency  of  use  and  the  rotation  with  other  active  substances.  For  instance,  for  products 
against  house  flies,  a  label  could  state  that  the  product  should  not  be  used  more  than  five 
times  per  year  and  should  only  be  used  in  rotation  with  at  least  one  other  product  with  a 
different mode of action. 

For consumer products  it is necessary to make clear that there might be a risk  of building up 
resistance and that this can be reduced. Since consumers have no knowledge of resistance the 
label claim should contain information to prevent it. For instance, the following sentence could 
be  added  to  the  label:  “When  the  product  is  not  used  according  to  the  label  resistance  of 
insects might occur. When the infestation persists contact a professional.” 

More information on resistance can be found in Chapter 6.2 of this TNG on Product Evaluation 
and the Insecticide Resistance Action Committee (IRAC: www.irac-online.org). 

5.6.4.1.4 Methodology of assessment 

Methods of application and dose rates 

When considering the overall evaluation of a proposed label claim competent authorities should 
ensure  that  the  data  presented  are  relevant  not  only  to  biological  challenge  and  treatment 
environment  but  also  that  the  method  of  application  and  application/dose  rate(s)  used  in  the 
test(s) are appropriate to the label claims and proposed use of the product. 

The  application  technique  should  therefore  reflect  the  claims  proposed  on  the  label,  whether 
crack and crevice, spot, space spray, contact spray or total release. 

General considerations 

The  efficacy  data  submitted  should  demonstrate  that  the  biocidal  product,  when  used  as 
directed  by  the  product  label,  will  result  in  a  measurable  beneficial  effect.  The  data  supplied 
should  demonstrate  that  an  acceptable,  consistent  level  and  duration  of  control  or  other 
intended effect will result from the use of the product at the recommended dose rate. 

This  may,  depending  on  the  individual  product,  be  measured  as  a  reduction  of  the  pest 
population  to  an  acceptable  level  or  a  reduction  in  damage.  The  acceptable  level  may  vary 
depending on the purpose of the proposed use. 

Competent authorities should evaluate available data to determine whether they are sufficient 
to support a label claim. 

 
 
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The  competent  authority  will  examine  the  submitted  data  package  and  a  judgment  will  be 
made  as  to  whether  any  data  omissions  are  considered  significant  as  to  delay  assessment. 
Those so identified will be communicated back to the applicant. The applicant can then supply 
additional data or modify their label claims in line with whatever has been supported. 

Any  known  limitations  on  efficacy  (including  resistance)  should  be  considered  during  the 
assessment. 

•  possible  restrictions  or  recommendations  concerning  the  use  of  the  product  in  specific 
environmental  or  other  conditions.  State  possible  factors  that  can  reduce  the  efficacy, 
for  instance  hot,  cold  or  humid  environments  or  the  presence  of  other  substances,  in 
addition to the grounds for these. Possible recommendations concerning the avoidance 
of  the  continuous  use  of  the  product  in  order  to  prevent  the  development  of  resistant 
strains and the grounds for these (see also TNsG on product authorisation Chapter 6.2). 
State if the product cannot be mixed with, for example, other biocidal products or if the 
use of the product with other biocidal products is recommended; 

• 

the  guidance  given  on  resistance  for  the  corresponding  data  requirement  of  the  active 
substance also applies here. 

5.6.4.1.4.1 Assessment of specific claims 

Sometimes a claim will include specific properties of the product, for instance: 

•  kills within 15 minutes; 

• 

• 

• 

residual effect up to 3 months; 

storage period up to 5 years; 

control of tropical ants. 

Where a particular property is claimed the data submitted to support the product should show 
that  the  product  actually  has  these  properties.  If  data  do  not  support  this  claim,  the  product 
may  still  gain  authorisation  with  amended  label  claims,  provided  that  the  product  still  shows 
acceptable efficacy. 

For  example:  If  a  product  claims  complete  control  of  ants  within  2  weeks  of  application,  the 
data submitted must show a high level of mortality (approximately 100%) within two weeks of 
application in order for these claims to be acceptable. 

However,  if  the  submitted  data  showed  90%  mortality  within  2  weeks  and  100%  mortality 
within 3 weeks, the product may still gain authorisation provided that the product claims were 
amended to ‘complete control of ants within 3 weeks of application’. 

Situations  such  as  the  example  above  will  require  each  study  to  be  evaluated  on  its  own 
merits,  taking  into  account  what  the  data  is  actually  showing.  Evaluators  must  use  scientific 
judgement to determine when authorisation would not be acceptable. 

For example: 

If a product  claims to kill ants within 15 minutes  of application, the data 
submitted must show sufficient mortality within 15 minutes of application 
in order for these claims to be acceptable. 

However,  if  the  submitted  data  showed  50%  mortality  within  15  minutes  but  90%  mortality 
within 2 hours, the product would still not be granted authorisation on the basis that for claims 
such  as  ‘kills  ants’,  the  average  user  would  expect  a  rapid  visual  effect  following  application 
(unless the product label clearly states how long the product takes to have an effect). 

5.6.4.1.5 Assessment of authorisation 

When considering the overall evaluation of proposed label claims, competent authorities should 
ensure that the data and the method of application and application/dose rates used in the tests 
are appropriate to the label claims and proposed use of the product. 

5.6.4.1.5.1 Norms and criteria 

The test results are compared directly with the norms and criteria for efficacy  described below 
per  insect/arthropod  pest.  The  performance  criteria  set  in  this  guidance  ask  for  high  levels  of 
efficacy,  which  is  of  course  what  we  aim  for.  However,  some  products  that  do  not  fully  meet 
the criteria can still be valuable in some cases. 

 
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When a product does not perform to the criteria it should be justified in the application why this 
product is still recommended for authorisation. For example, in a field trial the criteria may not 
be met because of immigration of insects from untreated areas (e.g. flies, mosquitoes). When 
this  is  explained  well  in  a  justification  the  product  might  still  be  accepted  for  authorisation, 
depending on the results of other field trials, simulated use and laboratory trials. 

Special attention  should be paid to  resistance, since under low insecticide pressure  resistance 
can build up more easily. Moreover, it should be taken care of that no placebo’s or misleading 
products  are  registered.  If  the  efficacy  level  is  significantly  lower  than  the  criteria  state  it 
should be mentioned on the label. 

The justification will be evaluated  case by  case. The product  should  not be authorised, unless 
there is a good reason for having a product of lower effectiveness. 

5.6.4.1.5.2 Assessment 

The  assessor/expert  assesses  on  the  basis  of  the  label  claim  and  the  above  criteria.  If  the 
product  was  assessed  to  be  sufficiently  effective  in  laboratory  and/or  field  tests,  it  will  be 
authorised as far as efficacy is concerned. 

5.6.4.2 General Claims: Crawling Insects, Flying Insects, Acaricide 

5.6.4.2.1 Introduction 

Some  products  have  a  very  broad  claim:  against  crawling  insects,  against  flying  insects, 
insecticide-acaricide spray, etc. In these cases it is not possible to test the product against all 
claimed  target  pests.  For  each  group  claimed  tests  should  be  performed  on  a  few  relevant 
species, of significant importance, and on the species specifically claimed on the label. 

General  claims  (e.g.  insecticide,  crawling  insects)  cannot  be  used  for  bait  products,  since  the 
bait differs per insect species. 

5.6.4.2.1.1 Crawling insects 

A  crawling  insect  is  defined  as  an  insect  that  generally  moves  on  the  ground.  These  include 
amongst  others  cockroaches,  ants,  fleas,  crickets,  silver  fish,  bed  bugs  and  carpet  beetle 
larvae.  The  effect  of  biocides  on  these  insects  is  primarily  based  upon  contact.  The  products 
involved  can  be  sprays,  dusts,  etc.  Amongst  the  crawling  insects,  cockroaches  are  the  most 
difficult to control. 

5.6.4.2.1.2 Flying insects 

A  flying  insect  is  defined  as  an  insect  that  generally  flies  from  one  spot  to  the  other.  These 
include  flies,  mosquitoes,  wasps  and  moths.  The  products  involved  can  be  sprays,  strips, 
paints, etc. 

5.6.4.2.1.3 Insecticide, acaricide and other arthropods 

A general claim for insecticides includes all insects. A general claim for acaricides includes ticks 
and  mites.  Other  arthropods  could  include  spiders  (Araneae),  harvestmen  (Opiliones), 
centipedes (Chilopoda), millipedes (Diplopoda), woodlice (Isopoda) and scorpions (Scorpiones). 

5.6.4.2.2 Dossier requirements 

A  clear  label  claim  should  be  submitted.  The  study  results  of  trials  should  demonstrate  the 
efficacy of the product based on the submitted label claim. Laboratory, simulated-use tests and 
field trials with the test organisms are needed to assess the efficacy of the product. Ideally, the 
studies  should  be  performed  according  to  established  guidelines  where  these  are  available. 
These may be international, EU or national guidelines. Ideally, data should be generated using 
national  or  international  recognised  testing  methods  (ISO,  CEN,  OECD,  etc.)  where  available 
and  appropriate.  See  Appendix  18  for  a  list  of  available  guidelines.  If  there  are  no  guidelines 
available  or  guidelines  are  not  suitable,  the  applicant  may  use  their  own  methods  (intra-
company Standard Operating Procedures), on condition however, that the study is scientifically 
robust,  well  reported,  provides  a  clear  answer  to  the  question  and  demonstrates  the  efficacy 
claimed.  In  addition,  the  test  methods  applied  and  the  test  conditions  should  be  clearly  and 
fully described and must address the efficacy claim that appears on the product label. A control 
treatment without biocide (negative control) should be included in all laboratory trials. 

 
 
 
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In  the  case  of  field  trials  where  true  replication  is  almost  certainly  impossible  to  achieve  and 
where  normal  control  methods  are  not  restricted  to  use  of  a  single  biocidal  product,  a  full 
description  of any factors that might be expected to influence product performance  should be 
given.  These  may  include  the  risk  of  re-invasion  from  adjacent  areas,  general  levels  of 
sanitation,  treatment  history,  season,  etc.  and  are  intended  to  provide  the  authorities  with 
information to assist with the interpretation of the results obtained. 

5.6.4.2.2.1 Test species 

Claim: crawling insects. In case of an application for authorisation of a product with a claim 
of “killing crawling insects” a product, which has demonstrated sufficient effectiveness against 
cockroaches,  may  also  be  authorised  to  control  other  crawling  insects.  However,  if  also 
population control and/or nest kill is claimed both cockroaches and ants have to be tested. 

Tests  with  cockroaches  should  normally  be  performed  with  two  key  species,  one  small,  one 
large,  such  as  the  German  cockroach  (Blattella  germanica)  and  either  the  oriental  cockroach 
(Blatta  orientalis)  or  the  American  cockroach  (Periplaneta  americana).  Tests  with  ants  should 
normally be performed with the Black garden ant (Lasius niger). 

Claim: flying insects. In case of an application for authorisation of a product with a claim of 
“killing  flying  insects”  tests  should  be  provided  with  flies,  mosquitoes  and  wasps.  Tests  with 
flies should normally be performed with the house fly, Musca domestica. Tests with mosquitoes 
should normally be performed with  Culex spp.  Test  with wasps should normally be performed 
with Vespula spp. 

Claim:  acaricide.  If  a  product  is  claimed  to  be  an  acaricide  tests  should  be  provided  with 
mites and ticks. What species should be used depends on the area of use (house dust mites in 
homes,  flour  mites  in  storage  rooms,  etc.,  for  instance:  Dermatophagoides  pteronyssinus, 
Tyrophagus  putrescentiae,  Acarus  siro).  For  mites  and  ticks  relevant  species  can  be  found  in 
sections 7 and 8. 

Claim:  other  arthropods.  For  this  claim  the  applicant  should  provide  information  on  what 
organisms are relevant for the intended use. At least some example should be given and these 
should be tested. 

Specific claim next to general claim: 

Whenever  efficacy  against  a  specific  organism  is  claimed  next  to  a  general  claim  or  as 
specification  of  a  general  claim  (e.g.  crawling  insects,  including  bedbugs),  tests  against  this 
organism should be provided. 

5.6.4.2.2.2 Laboratory tests and field trials 

Test  requirements  for  each  test  species  can  be  found  at  the  following  sections  dedicated  to 
these  insects/acarids.  For  other  arthropods  a  field  trial  should  be  provided  or  a  good 
justification why this is not appropriate. 

5.6.4.2.3 Assessment of authorisation 

5.6.4.2.3.1 Norms and criteria 

A  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient  level  of  efficacy”.  For 
products  with  general  claims  the  performance  criteria  per  tested  organism  are  the  same  as 
those for products with a specific claim for the test species. I.e. for crawling insects the criteria 
are the same as for cockroaches and ants, for flying insect the same as flies, mosquitoes and 
wasps, etc. The criteria can be found in the sections dedicated to these insects/acarids. 

5.6.4.3 Cockroaches 

5.6.4.3.1 Introduction 

Cockroaches are a common and persistent problem in many households. These crawling insects 
(although  several  species  can  also  fly)  are  scavengers  allowing  them  to  readily  adapt  to 
changing  food  availability.  Cockroaches  can  carry  bacteria  such  as  Salmonella  in  areas  co-
inhabited by humans. Cockroaches are also identified as a major cause of allergies and asthma, 
particularly  in  children.  Amongst  the  crawling  insects,  cockroaches  are  the  most  difficult  to 
control. 

 
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The  effect  of  biocides  on  these  insects  is  mainly  based  on  either  contact,  both  dermal  and 
tarsal, or the ingestion of bait products. 

5.6.4.3.1.1 Biology 

Cockroaches belong to the (sub) order Blattodea. There are over 3500 species of cockroaches, 
but  only  a  few  are  considered  domestic  pests  in  the  EU.  The  German  cockroach,  Blattella 
germanica, is the most common. 

Upon hatching from an egg capsule, cockroaches begin their nymphal stage (smaller version of 
adults  minus  fully  developed  wings  and  sex  reproduction  organs)  and  moult  through  various 
instars  until  reaching  the  adult  stage.  Time  of  development  can  take  weeks  or  months 
depending  upon  the  species  and  the  surrounding  environmental  conditions.  For  instance  the 
eggs  of  German  cockroaches  hatch  after  3  to  5  weeks  (depending  on  the  temperature),  the 
nymphal  stage  (5  to  7  moultings)  can  be  40  days  to  6  months  and  the  adults  live  about  6 
month (longer under lab conditions). 

In  temperate  European  countries  most  cockroach  species  will  almost  never  be  found  outside, 
with foraging activities almost entirely within human-made structures. 

5.6.4.3.2 Dossier requirements 

A clear label with comprehensive claims should be submitted. The study results of trials should 
demonstrate the efficacy of the product based on the submitted label claim. Requirements can 
differ for products for professional use and for consumer products. For professional use a field 
trial  is  always  required,  for  consumer  products  in  some  cases  laboratory  and  simulated-use 
tests are sufficient. If the product is applied as a bait, the entire bait (formulated, including the 
bait box if applicable) should be tested, not only the active substance which is contained in the 
bait. 

Ideally,  the  studies  should  be  performed  according  to  established  guidelines  where  these  are 
available. These may be international, EU or national guidelines. See Appendix  18 for a list of 
available guidelines. Appendix 19 gives an example of a test guideline that can be used. If the 
available  guidelines  are  not  suitable,  industry  standard  or  a  company’s  own  protocols  are 
acceptable,  on  condition  however,  that  the  study  is  scientifically  robust,  well  reported  and 
provides  a  clear  answer  to  the  question.  In  addition,  the  test  methods  applied  and  the  test 
conditions  should  be  clearly  and  fully  described  and  must  address  the  efficacy  claim  that 
appears on the product label. A control treatment without biocide (negative control) should be 
included in all laboratory trials. 

In  the  case  of  field  trials  where  true  replication  is  almost  certainly  impossible  to  achieve  and 
where  normal  control  methods  are  not  restricted  to  use  of  a  single  insecticidal  product,  a  full 
description  of  any  factor  that  might  be  expected  to  influence  product  performance  should  be 
given. These may include the  risk of re-invasion from adjacent areas, pest activity before the 
trial is initiated, general levels of sanitation, treatment history, season, etc. and are intended to 
provide the authorities with information to assist with the interpretation of the results obtained. 

5.6.4.3.2.1 Test species 

For use against cockroaches data against two key species, one small species normally German 
cockroach  (Blattella  germanica)  and  one  large  species  either  the  Oriental  cockroach  (Blatta 
orientalis)  or  the  American  cockroach  (P.  americana),  should  normally  be  available  for  spray 
products (aerosol, space spray, residual spray) to support general claims against cockroaches. 
For  bait  products,  the  label  can  only  claim  efficacy  against  species  that  have  been  treated 
under field conditions. 

5.6.4.3.2.2 Laboratory tests and field trials 

For the evaluation of biocides against cockroaches different types of laboratory, simulated-use 
tests and field tests can be used. Examples of test are listed below. 

Screening Studies (No- Choice Test) 

The  product  is  applied  to  representative  surfaces  or  via  direct  cuticle  application,  in  an  arena 
with  cockroaches,  to  assess  inherent  contact  toxicity  or  knockdown  effects  of  the  active 
substance. Specify whether adults (male or female) or nymphs are used. Tests may be used to 
demonstrate  basic  efficacy  or  efficacy  against  insects,  resistance  to  specific  chemicals  (LD50 

 
 
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versus  a  susceptible  field  strain)  or  insect  growth  regulator  effects  (nymphs  are  treated  and 
subsequent effects are recorded such as inhibition of moulting, deformities, sterile adults). 

Results support descriptions related to the mode of action (symptomology) or “effective against 
strains resistant to “x” class of insecticides”, or similar efficacy claims. 

For  bait  products  dietary  bioassay  studies  can  be  conducted  using  the  biocidal  bait  as  a  food 
source. Replicate groups of test insects are exposed to either a continuous toxic diet, or a toxic 
diet for 24 hours and then a non-toxic diet for the rest of test period. 

In all laboratory studies a treatment without biocide should be conducted as a negative control, 
with insects from the same insect population and with the same number of replicates. 

Screening  tests  are  not  always  necessary.  When  efficacy  is  demonstrated  in  residual  tests, 
palatability  tests  or  similar  tests,  this  is  deemed  sufficient.  Screening  tests  can  sometimes  be 
used as bridging studies: if tests involving a product result in similar effects in different target 
species, field studies can be waived for some insect species. 

Determination of residual efficacy 

Formulated  product  (spray,  powder,  dust,  etc.)  is  applied  to  representative  surfaces  at  a 
specified  dose  rate,  or  rates,  including  the  recommended  label  rate(s).  Cockroaches  (adults) 
are  exposed  to  the  deposit  at  several  time  intervals  after  application  (including  the  day  of 
treatment and at the end of the claimed residual period). Exposure time should, preferably, be 
comparable to the time the cockroaches might reasonably be expected to be in contact with a 
treated  surface  under  natural  conditions  (e.g.  10  min  -  1  hour)  and  assessors  will  take  this 
factor into consideration when evaluating the data. Treated surfaces should include at least one 
porous  and  one  non-porous  substrate  (or  according  to  the  label  claim)  representing  surfaces 
that  might,  typically,  be  treated  for  cockroach  control  (e.g.  ceramic  tile,  plywood,  painted 
plywood, stainless steel, concrete). Mortality is normally assessed 1 day and up to 7 days post-
exposure. 

To  substantiate  a  knockdown  claim  the  number  of  cockroaches  on  their  backs  is  counted  at 
stated times after exposure (typically at 5 minute intervals until +30 min, then again at 45 and 
60  min).  The  time  until  50%  (KT50)  and  95%  (KT95)  of  the  insects  are  knocked  down  is 
derived statistically. 

For insect growth  regulators, exposure  conditions can be as described above, but selection  of 
the developmental stage (nymph, adult) and post-exposure assessment (deformities, moulting 
success,  sterility,  mortality)  must  be  adapted  to  suit  the  mode  of  action  of  the  active 
substance. Hence, assessments may continue to be made several weeks after exposure (sub-
lethal  or  non-lethal  effects  on  fertility,  sterility  for  example  may  contribute  to  long  term 
population control without short term mortality). 

Groups  of  cockroaches  of  the  target  species  should  be  of  specified  age/sex  and  number. 
Normally  tests  are  performed  with  5  or  more  replicates,  with  at  least  10  cockroaches  per 
replicate.  When  only  3  replicates  are  used,  at  least  20  insects  per  replicate  should  be  used. 
Replicates should be conducted per applied dose, time point, surface, and a reference product 
(at registered rate) and untreated surfaces should be included as negative controls. 

Environmental conditions must be specified for the test itself, and during storage of the treated 
substrates (temperature, humidity, photoperiod). Temperature would be expected to fall in the 
range  19-29°C.  When  efficacy  at  high  temperatures  is  claimed  40°C  would  be  a  good  test 
temperature. 

Palatability tests with bait products 

The aim of the bait choice feeding trials is to determine the palatability of the product for the 
test  insect.  If  conducted  on  both  fresh  and  aged  product  it  may  provide  information  on  the 
storage stability of the product. In this test design, nymphs and adults of German and Oriental 
cockroaches have the choice between a non-toxic food source (challenge diet, either the non-
toxic bait or a non-toxic food source known to be a strong feeding source for the test species) 
and  the  bait  containing  the  active  substance.  Normally  tests  are  performed  with  5  or  more 
replicate  tests,  with  at  least  10  cockroaches  per  replicate.  When  only  3  replicates  are 
employed, at least 20 insects per replicate should be used. In all laboratory studies a treatment 
without  biocide  should  be  conducted  with  insects  from  the  same  insect  population,  as  a 
negative control. 

 
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The  test  should  demonstrate  acceptable  toxicity  in  competition  with  the  alternative  food 
source. 

The  population  composition  (males,  gravid  non-gravid  females,  nymphs)  in  these  tests  is  of 
importance.  Preferably  mature  insects  should  be  used  since  immature  stages  do  not  need  to 
feed  every  24  hours.  It  should  be  noted  that  the  feeding  behaviour  of  German  cockroach 
females,  changes  during  ‘pregnancy’  and  that  early  instar  nymphs  tend  to  forage  less  than 
older instars. 

Simulated use 

These tests are designed to mimic the practical use situation. The insects must have a choice 
to be in contact with the biocide or not. For example, cockroaches (B. orientalis, B. germanica) 
can be introduced into choice boxes with one half of the base surface being sprayed with a test 
formulation.  Food  and  water  is  always  on  the  non-treated  area  to  be  reached  by  the  animals 
without crossing the treated area. Variations on this test would be to expose insects (voluntary 
contact)  to  a  variety  of  different  treated  surfaces,  e.g.  plywood,  cement,  vinyl,  ceramic  tiles, 
glass etc. 

For  products  claiming  “population  control”  (eradicates  cockroach  population)  an  entire 
population or at least different life stages should be tested while there is a possibility that only 
a few individuals get in contact with the biocide. 

For “secondary kill” (kills cockroaches that do not visit the bait, however, not always the whole 
population)  claims  at  least  different  life  stages  should  normally  be  tested  where  only  a  few 
individuals get in contact with the biocide directly. Life stage is dependent on a specific mode of 
action (necrophagy versus coprophagy) and the claim. Either nymphs or adults could be used. 

In all laboratory studies a treatment without biocide should be conducted with insects from the 
same insect population, as a negative control. 

Field trial 

In field trials the product is tested in actual use situation, for instance in an infested home or 
warehouse  and  applied  according  to  the  direction  for  use  on  the  label.  An  example  of  the 
results to be achieved in a field trial can be found in Appendix 19. 

5.6.4.3.2.3 Requirements per type of claim 

Per type of claim the requirements will be listed. 

Products intended for use as general surface treatment or aerosol for consumers: 

•  a  laboratory  test  showing  mortality  and/or  knockdown  and/or  residual  efficacy, 

depending on the claim; 

•  a simulated-use test showing mortality and knockdown according to the claim. 

Products intended for use as general surface treatment or aerosol for professionals: 

•  a  laboratory  test  showing  mortality  and/or  knockdown  and/or  residual  efficacy, 

depending on the claim; 

•  a simulated-use test showing mortality and knockdown according to the claim; 

•  a field trial according to the directions for use. 

Products  intended  for  use  as  general  surface  treatment  or  aerosol  with  a  claim  of  population 
control or secondary kill: 

•  a laboratory test showing residual efficacy; 

•  a simulated-use test showing mortality according to the claim; 

•  a field trial according to the directions for use. 

Products intended for use as baits: 

•  due to the specificity of baits, only effects against species of cockroach that have been 

tested in the field can be claimed on the product label; 

•  a  laboratory  test  showing  palatability,  of  fresh  product  and  product  at  the  end  of  the 

claimed maximum storage period; 

•  a simulated-use test showing mortality according to the claim; 

•  a field trial according to the directions for use and with the claimed cockroach species. 

 
 
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Simulated-use tests can be waived if a robust field trial is submitted. 

5.6.4.3.3 Assessment of authorisation 

5.6.4.3.3.1 Norms and criteria 

A biocidal product may only be authorised if it “possesses a sufficient level of efficacy” (BPD). 
This is implemented in the following way. 

An  insecticidal  product  intended  for  the  control  of  cockroaches  is  normally  considered  to  be 
sufficiently “effective” if the following results can be achieved: 

Products intended for use as general surface treatment or aerosol for consumers: 

• 

required results in laboratory tests and simulated-use tests:  
o  ≥ 90% knockdown within a few minutes after contact with the product (or according 

to the claim), direct after spray and at the end of the residual period claimed; 

o  mortality according to the label claim, preferably ≥90% in 24 hour. 

Products intended for use as general surface treatment or aerosol for professionals: 

• 

required results in laboratory tests: 

o  direct  application:  100%  mortality  within  1  hour  after  spraying  the  cockroaches, 
mortality between 90 and 100% can be accepted provided a qualified explanation is 
given for the lack of total control; 

o  residual  test:  100%  mortality  within  24  hours  after  placing  the  cockroaches  in  the 
test  area, direct after spray and at the end of  the claimed  residual period. Mortality 
between 90 and 100% can be accepted provided a qualified explanation is given for 
the lack of total control. 

• 

required results in field test: 

o  after  a  period  of  2-10  weeks,  the  population  reduction  exceeds  ≥90%  relative  to 
either  untreated  sites  or  pre-treatment  levels.  If  retreatment  is  necessary  100% 
mortality should then be achieved. 

Products  intended  for  use  as  general  surface  treatment  or  aerosol  with  a  claim  of  population 
control or secondary kill: 

• 

• 

required results in laboratory tests and simulated-use tests: 
o  ≥  90%  mortality  within  the  test  period,  direct  after  spray  and  at  the  end  of  the 

residual period claimed; 

required results in field tests: 
o  after a period of 2-10 weeks, the population reduction exceeds 90% relative to either 

untreated sites or pre-treatment levels. 

Products intended for use as baits: 

• 

• 

• 

required results in laboratory palatability choice test (bait and alternative food): 
o  at least 95% of the test insects have been killed at a given time point; 

required results in simulated-use tests: 
o  ≥ 90% reduction of the population within a few weeks; 

required results in field tests: 
o  after a period of 2-10 weeks, the population reduction exceeds 80% relative to either 

untreated sites or pre-treatment levels. 

Products based in insect growth regulators (IGR): 

• 

required results in laboratory tests: 

o  at least 95% of the insects does not develop to the next instar; 

• 

required results in simulated-use tests: 
o  ≥ 90% reduction of the population within a few weeks; 

• 

required results in field tests: 

o  after  a  period  of  6  -14  weeks,  the  population  reduction  exceeds  80%  relative  to 

either untreated sites or pre-treatment levels. 

Deviation from these norms is possible but should be justified in the application. 

 
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Field trial data at the label application rate(s) must preferably be evaluated by an experienced 
assessor  since  performance  can  vary  considerably,  even  from  apartment  to  apartment  in  the 
same building. Number of trials, the complexity of the trials sites, the use (or not) of additional 
measures  that  can  contribute  to  effective  control,  treatment  history,  etc.  can  all  have  a 
substantial effect upon the level of control that is achieved. The data must provide evidence of 
suitable  levels  of  efficacy  during  the  residual  period  claimed,  relative  to  pre-treatment 
population  assessments  and/or  performance  of  reference  products  under  similar  conditions, 
and/or  assessments  of  cockroach  populations  in  untreated  areas  under  similar  conditions. 
Where  mean  population  reduction  exceeds  90%  relative  to  either  untreated  sites  or  pre-
treatment  levels,  the  product  is  considered  effective,  but  the  assessor  has  the  discretion  to 
view  each  data  set  on  its  merits  and  consider  all  factors  before  concluding  whether  the  data 
support the claimed level of performance or not. 

5.6.4.4 Ants 

5.6.4.4.1 Introduction 

Ants may cause inconvenience both indoors and outdoors. 

In Europe the following ant species are common: 

Black garden ant,  
Pavement ant 
Red ant 
Erratic ant 

Lasius spp., most common L. niger 
Tetramorium caespitum  
Myrmica rubra 
Tapinoma erraticum. 

Next to these native ant species tropical ants can cause inconvenience, mainly indoors.  

Of  the  tropical  ant  species  there  are  two  species  that  are  most  commonly  found  causing 
inconvenience in buildings in Europe: 

Pharaoh ant 
Argentine ant 

Monomorium pharaonis 
Linepithema humile. 

5.6.4.4.1.1 Biology 

Ant  development  involves  a  complete  metamorphosis  that  includes  distinct  egg,  larval,  pupal 
and  adult  stages.  Most  ant  species  form  colonies  comprised  of  complicated  social  structures 
that  include  infertile  female  workers,  one  or  more  specialised  fertile  queens  and  (at  certain 
stages in nest development) sexually mature  males. Some  species have developed additional 
specialised  workers  that  are  responsible  for  guarding  the  nest  and  attacking  intruders,  whilst 
others  perform  domestic  and  foraging  duties.  These  workers  will  actively  forage  on  a  wide 
range  of  foods  including  sweet  substances,  seeds,  insects  and  aphid  secretions.  A  successful 
foraging  ant  also  has  the  ability  to  communicate  where  to  find  food  to  her  co-workers,  using 
chemical signals (trail pheromones). 

5.6.4.4.2 Dossier requirements 

A clear label claim should be submitted. The study results of field trials should demonstrate the 
efficacy of the product, based on the submitted label claim. 

Requirements  can  differ  for  products  for  professional  use  and  for  consumer  products.  For 
professional  use  products  a  field  trial  is  always  required,  while  laboratory  and  simulated  use 
tests  might  be  considered  sufficient  in  some  cases  for  consumer  products.  Requirements  also 
depend on the use: for “nest kill” and bait products alike, both laboratory and field trials with 
the  test  insects  are  needed;  for  products  that  only  claim  to  kill  individual  insects  that  are  in 
contact  with  the  biocide,  laboratory  and  simulated-use  tests  are  sufficient.  If  the  product  is 
applied  as  a  bait,  the  entire  bait  (formulated,  including  the  bait  box  if  applicable)  should  be 
tested, not only the active substance which is contained in the bait. 

Ideally,  the  studies  should  be  performed  according  to  established  guidelines  where  these  are 
available. These may be international, EU or national guidelines. See Appendix  18 for a list of 
available guidelines. 

If  there  are  no  guidelines  available  or  the  guidelines  are  not  suitable,  the  applicant  may  use 
their  own  methods  (intra-company  Standard  Operating  Procedures),  on  condition  however, 
that  the  study  is  scientifically  robust,  well  reported  and  provides  a  clear  answer  to  the 
question.  In  addition,  the  test  methods  applied  and  the  test  conditions  should  be  clearly  and 

 
 
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fully  described  and  must  address  the  efficacy  claim  that  appears  on  the  product  label.  In  the 
case  of  field  trials  where  true  replication  is  almost  certainly  impossible  to  achieve  and  where 
normal  control  methods  are  not  restricted  to  the  use  of  a  single  insecticidal  product,  a  full 
description  of any factors that might be expected to influence product performance  should be 
given.  These  may  include  the  risk  of  re-invasion  from  adjacent  areas,  general  levels  of 
sanitation, treatment history etc. and are intended to provide the authorities with information 
to assist with the interpretation of the results obtained. 

5.6.4.4.2.1 Test species 

Table  26  below  shows  for  this  group  of  insecticides  the  possible  combinations  of  target 
organisms, and the corresponding test organisms on which efficacy is tested in both laboratory 
and field tests. The selection of test species should be relevant to the label claim. 

Table 26: Target organisms versus test organisms 

Target organisms of the 
insecticide: 

Test organisms: 

Ants 

Tropical ants 

Garden ant (Lasius niger) 

Pharaoh ant (Monomorium pharaonis), 
Argentine ant (Linepithema humile) 

5.6.4.4.2.2 Laboratory tests and field trials 

For  the  evaluation  of  biocides  against  ants  different  types  of  laboratory,  simulated-use  tests 
and field tests can be used. 

Screening studies for direct spray or general surface treatments 

In all laboratory studies a treatment without biocide should be conducted with insects from the 
same insect population, as a negative control. Examples of tests are: 

Direct spray: 20 ants placed within a Petri dish and directly sprayed with material. Knockdown, 
time to death and total mortality is recorded. For insecticides with a “nest kill” claim the time to 
death will be longer (>1 day) since these ants have to live long enough to take the insecticide 
into  the  nest.  Normally  at  least  5  replications  and  5  non-treated  controls  should  be  used. 
Controls are very important in this case, as it often turns out to be very difficult to keep ants 
active in trials. 

Residual  spray:  20  ants  placed  on  a  surface  treated  with  the  product.  Ants  are  placed  in  the 
arena directly after application, at several time intervals after application and also at the end of 
the  period  claimed  for  residual  effect.  The  time  to  death  of  the  ants  and  total  mortality  is 
recorded. 

A control treatment without biocide should be included in all laboratory trials. Normally at least 
5 replications and 5 non-treated controls should be used. 

Palatability tests with bait products 

The important factors relating to testing bait products are to establish the appropriate dosage 
and  intrinsic  palatability  of  the  formulation  in  laboratory  tests.  Claims  made  for  bait  products 
should  distinguish  between  ants  and  tropical  ants,  since  the  latter  can  be  attracted  by 
completely  different  baits  than  the  more  common  European  ant,  L.  niger.  Data  should  be 
provided for all species, for which claims are made. 

The most important factor involved in laboratory testing is to provide a free choice alternative 
food  source  to  the  test  insects.  This  may  be  sugar-based  materials  for  European  ants  and 
protein-based  materials  (meat,  eggs,  dead  insects)  for  some  tropical  ants.  The  formulation 
should  demonstrate  acceptable  toxicity  in  competition  with  the  alternative  food  source.  A 
control treatment without biocide of similar  size as the test itself (i.e. number of replications) 
should be included in all laboratory trials. 

When a product is claimed to be effective after a long period of storage, it is also necessary to 
demonstrate  that  the  product  will  still  be  effective,  and  attractive,  after  the  stated  storage 
period. The applicant must either provide data on the palatability of the product at the end of 
maximum storage period or alternatively (in case of a new product) data gained in a stress test 

 
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with  'accelerated  ageing',  i.e.  a  palatability  test  with  the  product  which  is  stored  under 
challenging conditions. 

Simulated use studies 

These tests are designed to mimic the practical use situation.  The tests should be relevant to 
the  use  and  label  claims.  A  control  treatment  without  biocide  should  be  included  in  all 
laboratory tests. Control trials should be of similar size (i.e. number of replications) as the test 
itself, to make statistical comparison possible and to get a fair impression of control mortality. 

Examples of tests are: 

Direct general surface treatments without nest kill: 
Ants  (normally  at  least  20  worker  ants)  can  be  introduced  into  choice  boxes/arenas  with  one 
half  of  the  base  surface  being  sprayed  with  a  test  formulation,  at  the  correct  application  rate 
according to the product label. Food and water is always on the non-treated area to be reached 
by  the  animals  without  crossing  the  treated  area.  Variations  on  this  test  would  be  to  expose 
insects  (voluntary  contact)  to  a  variety  of  different  treated  surfaces,  e.g.  plywood,  cement, 
vinyl, ceramic tiles, glass etc. Mortality is recorded. 

Normally tests should be performed in triplicate.  

Direct general surface treatments with nest kill:  
In  a  double  chamber  trial  an  ant’s  nest  (normally  at  least  20  (worker)  ants)  is  placed  within 
one  arena,  which  is  connected  to  another  arena.  Part  of  the  second  arena  is  treated  with  the 
insecticide  at  the  correct  application  rate  according  to  the  product  label.  Adequate  food  and 
water is placed on the non-treated surface of this second arena. Ants must be able to reach the 
food  without  contacting  the  treated  surface.  Normally  tests  should  be  performed  in  triplicate. 
Efficacy is assessed e.g. length of time taken to result in control of the ant population (e.g. no 
foraging ants). 

The  nest  should  be  opened  at  the  end  of  the  trial  (e.g.  1  week),  to  check  whether  all  ants 
within the nest are dead, especially the queen(s). 

Bait products: 
The efficacy of the entire formulated bait is tested, hence not only the active component within 
the  bait.  An  ant’s  nest  is  placed  within  an  arena  trial  under  controlled  conditions  (e.g.  with 
respect to temperature, relative humidity, photoperiod, etc.). Adequate food (bait without the 
active  substance  or  and  alternative  food  source)  and  water  are  placed  opposite  the  nest. 
Insects  are  allowed  to  acclimatise  for  7  days  before  introduction  of  bait.  An  additional  fasting 
period of 4 days, providing them with water only, is recommended. At regular time intervals (in 
hours),  the  attractiveness  of  the  bait  for  the  ants  is  recorded  (by  observing  whether  they 
approach the bait or avoid it). Ant mortality is recorded at regular time intervals (in days). At 
the  end  of  the  trial  the  nest  could  be  opened  to  check  whether  all  ants  within  the  nest, 
including the queen(s), are dead. 

Field trials for all claims 

The  tests  should  be  relevant  to  the  use  and  label  claims.  Tests  with  Lasius  niger  are  done 
preferably  during  the  early  spring.  In  the  end  of  summer  population  decline  might  be  due  to 
natural  causes  instead  of  the  insecticide.  Non-treated  nests  should  be  used  as  a  negative 
control, to test nest activity. 

Monitor  ant  numbers  at  various  locations  around  a  building  and  locate  the  entrances  of  nests 
and “ant-trails” (routes taken by ants). Apply the insecticide according to the label instructions. 

The efficacy tests against ants should normally be performed in a minimum of three objects. An 
object can be a place in or near the house, where ants cause inconvenience for the inhabitants. 
This may be in a house, on a balcony, a terrace or in a garden, depending on the field of use of 
the  product.  If  the  test  is  performed  outdoors,  records  of  temperature  and  rainfall  should  be 
kept. 

Monitoring  should  be  conducted  at  the  same  locations  (as  the  pre-treatment)  and  at  similar 
times during the entire trial (e.g. at 12.30, 13.00, etc.). Monitoring should continue (e.g. 1 day 
after treatment, 1 week after treatment, etc. at least once weekly) until control is seen. If no 

 
 
 
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ants are seen during a post-treatment monitoring visit then the site should be re-visited once 
to ensure that re-infestation does not occur. 

The  effect  on  the  ant  population  can  be  determined  by  counting.  For  this  purpose,  a  fixed 
position on the ‘ant-trail’ is to be used and a count of the number of any ants that pass is made 
in 1 minute, at several time intervals during the test. 

5.6.4.4.2.3 Requirements per type of claim 

Per type of claim the requirements will be listed. 

Products intended for use as general surface treatment for consumers: 

•  a  laboratory  test  showing  mortality  and/or  knockdown  and/or  residual  efficacy, 

depending on the claim; 

•  a simulated-use test showing mortality and knockdown according to the claim. 

Products intended for use as general surface treatment for professionals: 

•  a  laboratory  test  showing  mortality  and/or  knockdown  and/or  residual  efficacy, 

depending on the claim; 

•  a simulated-use test showing mortality and knockdown; 

•  a field trial according to the directions for use. 

Products intended for use as general surface treatment with a claim of nest kill:  

•  a laboratory test showing residual efficacy; 

•  a simulated-use test showing mortality;  

•  a field trial according to the directions for use. 

Products intended for use as baits: 

a)  Due to the specificity of baits, only effects against ant species that have been tested in 

the field can be claimed on the product label; 

b)  a laboratory test showing palatability; 

c)  a simulated-use test showing mortality; 

d)  a field trial according to the directions for use and with the claimed ant species. 

Simulated-use tests can be waived if a robust field trial is submitted. 

5.6.4.4.3 Assessment of authorisation 

5.6.4.4.3.1 Norms and criteria 

A biocidal product may only be authorised if it “possesses a sufficient level of efficacy” (BPD). 
This is implemented for ants in the following way. 

An  insecticide  against  ants  is  normally  considered  to  be  sufficiently  “effective”  if  the  following 
results can be achieved: 

Products intended for use as general surface treatment for consumers: 

• 

required results in laboratory mortality tests and simulated-use tests: 
o  ≥ 90% knockdown in 5 -10 minutes (or according to the claim), direct after spraying 

the ants and at the end of the residual period; 

o  mortality according to the label claim, preferably ≥90% after 24 hour. 

Products intended for use as general surface treatment for professionals: 

• 

required results in laboratory tests: 

o  direct application: 100% mortality within 24 hours after spraying the ants, mortality 
between 90 and 100% can be accepted provided a qualified explanation is given for 
the lack of total control; 

o  residual  tests:  ≥  90%  mortality  within  24  hours  after  placing  the  ants  in  the  test 

area, direct after spray and at the end of the residual period; 

• 

required results in field tests: 
o  after a period of 2-8 weeks, the population reduction exceeds 90% relative to  either 

untreated sites or pre-treatment levels. 

 
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Products intended for use as general surface treatment with a claim of nest kill:  

• 

• 

• 

laboratory tests: 
o  100%  mortality  within  the  test  period,  direct  after  spray  and  at  the  end  of  the 

residual period; 

required results in simulated-use tests: 
o  slow knockdown, ants must be able to reach the nest; 
o  ≥ 90% mortality within the test period, including ants in the nest; 

required results in field tests: 
o  after  a  period  of  2-8  weeks,  the  population  reduction  100%  relative  to  either 
untreated  sites  or  pre-treatment  levels,  in  case  of  lower  efficacy  it  has  to  be  shown 
that the queen(s) in the test nests is killed. 

Products intended for use as baits: 

• 

• 

• 

required results in laboratory palatability choice test (bait and alternative food): 
o  at least 95% of the test insects have been killed at a given time point; 

required results in simulated-use tests: 
o  ≥ 90% reduction of the population within a few weeks; 

required results in field tests: 
o  after a period of 2-4 weeks, the population reduction exceeds 90% relative to either 

untreated sites or pre-treatment levels. 

Deviations from these norms is possible but should be justified in the application. 

5.6.4.5 Termites 

5.6.4.5.1 Introduction 

Termites, in natural settings, work as beneficial insects by breaking down cellulose-containing 
materials, such as dead trees.  However, termites can cause damage to living trees and many 
crop  plants,  but  the  fact  that  they  can  use  dead  wood  makes  them  a  major  pest  for  timber 
used  both  outdoors  and  inside  buildings.  Termites  become  a  problem  to  humans  when  they 
infest  timber  used  in  constructions  (i.e.  wood  structures)  in  risk  areas.  Owing  to  their  high 
moisture  requirements,  they  usually  nest  in  soils,  but  can  invade  buildings  from  underneath 
through cracks and seams or by building shelter tubes connecting the wood to their nest in the 
soil.  In  Europe  and  in  the  European  tropical  overseas  regions,  there  are  three  main  types  of 
termites:  subterranean,  tree  and  drywood  termites,  the  subterranean  being  the  most 
destructive  termites  in  construction.  Due  to  their  biological  characteristics  (subterranean 
termites),  they  live  in  the  soil  and  must  maintain  contact  with  the  ground  or  some  other 
moisture source to survive. 

Insecticides  against  termites  can  be  divided  into  PT8  products,  preventive  treatments  to 
protect  the  wood  and  curative  treatments  on  the  wood,  and  PT18  products,  which  are 
considered in this section. 

5.6.4.5.1.1 Biology 

Termites  belong  to  the  order  of  Isoptera.  In  Europe  and  in  the  European  tropical  overseas 
regions  there  are  three  main  termite  families;  subterranean  (Rhinotermitidae),  drywood 
termites (Kalotermitidae) and tree termites (Nasutitermitidae). 

Reticulitermes  is  the  most  common  genus  encountered  from  the  Rhinotermitidae  family  in 
Europe.  The  main  species  registered  are:  R.  flavipes  (former  R.  santonensis),  R.  lucifugus,  R. 
lucifugus corsicus, R. grassei, R. banyulensis, R. balkanensis. 

They  are  widespread  around  the  Mediterranean  (Spain,  France,  Italy,  Portugal,  Balkans,  and 
Greece) and Black Sea (Turkey, Rumania), though some termite spots in the UK and Germany 
have been reported. Several unanswered questions remain about the origin of these termites. 
While some Reticulitermes are native to Europe, others may be related to species from eastern 
North America and the Middle East (Israel, Asian Turkey, etc.). 

Coptotermes  sp.  and  Heterotermes  sp.  are  the  main  two  species  belonging  to  the 
Rhinotermitidae family found in European tropical overseas regions. 

 
 
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Nasutitermes  sp.  are  the  main  species  belonging  to  the  Termitidae  family  (tree  termites) 
encountered in the European tropical overseas regions. 

Kalotermes  flavicollis  and  Cryptotermes  brevis  are  the  main  two  species  of  drywood  termites 
present  in  Europe  (especially  in  the  coastal  areas  of  Mediterranean  countries  and  Canary 
Islands). Cryptotermes sp. is a main genus belonging to drywood termites encountered in the 
European tropical overseas regions. 

A  brief  explanation  of  the  life  cycle  (figure  11)  may  help  to  clarify  the  difficulties  involved  in 
control  of  termites.  There  is  a  split  after  the  larval  stages  into  two  lines,  the  sexual  and  the 
worker  line.  Individuals  going  down  the  sexual  line  develop  into  nymphs  and  then  into  either 
alates  (which  are  the  reproductive  form  most  people  are  familiar  with)  or  neotenics 
(supplementary reproductives). The alates do form queens (physogastrics), however, these are 
much  more  mobile  than  those  found  in  tropical  species.  The  alternative  line  of  development, 
the  neutral  line,  is  the  development  of  larvae  into  workers,  which  in  turn  can  either  remain 
workers or develop into neotenics or soldiers. Workers are approximately 4 to 6 mm in length. 
An important feature in the biology of termites that makes them very difficult to control is the 
ability of individuals in both lines to form sexual reproductives and, hence, give rise to a new, 
viable  colony.  In  addition,  supplementary  secondary  reproductives  can  be  produced  in  very 
large numbers. 

Figure 11: Life cycle of subterranean termites 

5.6.4.5.1.2 Control methods 

Preventive treatments 

Traditionally, the methods used to fight termites were based upon treating infested or exposed 
wood  with  wood  preservatives.  This  is  valid  for  all  termite  types  (subterranean,  tree  and 
drywood). Those products are included in product type 8 (wood preservatives) of the BPD, and 
are not considered in this section. 

In  addition  to  the  preventive  treatment  of  timber,  a  barrier  can  be  used  to  isolate  the  paths 
used  by  subterranean  termites  to  access  the  building  from  underneath  where  the  nest  is 
located.  Barriers  systems  usually  consist  of  a  polymer  membrane  or  other  material  and  an 
insecticide (product type 18). The system is installed between the soil and the construction to 
keep  subterranean  termites  outside  and  to  eliminate  those  that  come  into  contact  with  the 
insecticide. 

Remedial treatments 

Different methods are currently used in Europe: 

Chemical barriers 
Methods based on treating the infested wood with wood preservatives are included in product 
type 8 (wood preservative) of the BPD, and are not considered in this section. 

In addition to the wood treatment, two types of chemical barriers are used to impregnate the 
walls of the construction and the soil around. 

egglarvanymphalateneotenicneotenicsoldierworker 
 
 
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Considering  the  subterranean  termites,  this  method  aims  to  eliminate  insects  inside  the 
construction and to protect it for several years. This method does not eliminate the nest (which 
is located in the soil). 

Bait system 
It consists typically of a cellulose-based matrix treated with a slow acting insecticide, which is 
consumed by workers and is spread through the colony by trophallaxis (one individual is fed by 
another). Consequently, this method may be useful to eradicate the whole colony. 

Treatment of waste 

In order to prevent termite contamination by waste infested and transported into an area not 
infested, it could be relevant to treat the waste with biocidal products. 

5.6.4.5.2 Dossier requirements 

A clear label claim should be submitted. 

Laboratory  and  field  trials  with  termites  are  needed  to  assess  the  efficacy  of  the  products. 
Ideally,  the  studies  should  be  performed  according  to  established  guidelines  where  these  are 
available.  These  may  be  EU  or  national  guidelines.  European  standardisation  work  is  being 
conducted  by  several  termite  experts  in  Europe.  At  this  moment,  no  European  standard  has 
been  published  yet,  only  French  standards  are  available.  However,  due  to  the  greater 
significance  of  termites  as  structural  pests  in  countries  outside  Europe,  such  as  the  United 
States and Australia, a variety of standard test methods are published, together with extensive 
reports in the scientific literature which may prove useful references. Account should be taken 
of results obtained using such methods, especially where the same termite species are present 
as  those  in  Europe  including  the  French  overseas  territories.  See  Appendix  18  for  a  list  of 
available guidelines (guidelines outside EU not included yet). 

If  there  are  no  guidelines  available  or  guidelines  are  not  suitable  to  evaluate  the  termiticide 
(e.g.  if  new  products  are  developed),  the  applicant  may  use  their  own  methods,  on  condition 
however, that the study is scientifically robust, well reported and provides a clear answer to the 
question.  In  addition,  the  test  methods  applied  and  the  test  conditions  should  be  clearly  and 
fully  described  and  must  address  the  efficacy  claim  that  appears  on  the  product  label.  In  the 
case of field trials a full description of any factors that might be expected to influence product 
performance  should  be  given.  These  may  include  the  risk  of  re-invasion  from  adjacent  areas, 
treatment  history,  etc.  and  are  intended  to  provide  the  authorities  with  information  to  assist 
with the interpretation of the results obtained. 

A  control  treatment  without  biocide  should  be  included  when  testing  any  termite  products  in 
laboratory trials. 

5.6.4.5.2.1 Test species 

A  product  against  termites  in  Europe  should  normally  be  tested  on  termites  belonging  to  the 
genus Reticulitermes. 

For European tropical overseas regions, the product should normally be tested at least against 
termites belonging to the genus Coptotermes and on every genus claimed by the applicant. 

Remarks: 

e)  In any case, the termite species needs to be identified and all useful information about 
the  colony  collected  (locality  of  origin,  laboratory  rearing  conditions,  characteristics  of 
their natural environment if termites are collected in field); 

f)  For the evaluation of termite baits, the species referred to in the label claims should be 
used.  If  the  claim  refers  generally  to  Reticulitermes  species  (without  specifying  the 
species), it is recommended to test, at least, two different European species in lab tests; 

g)  Due  to  the  specificity  of  baits,  only  effects  against  species  of  termites  that  have  been 

tested should be claimed on the product label. 

5.6.4.5.2.2 Laboratory tests and field trials 

The tests specified below are mainly for bait products. While laboratory tests can be conducted 
for  all  the  termiticide  products,  field  tests  are  addressed  specially  for  bait  products.  For 

 
 
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soil/wall  barrier  products  and  for  physico-chemical  systems  the  tests  should  be  designed  to 
mimic the practical use situation. The test should be performed according to the label claim. 

Due  to  the  specificity  of  baits,  only  effects  against  species  of  termites  that  have  been  tested 
should be claimed on the product label. 

The important factors relating to testing bait products are to: 

a)  establish the appropriate dosage of the formulation in laboratory tests. This can be done 
in  a  mortality  test  (evaluation  of  the  toxicity  of  the  insecticidal  formulation  in  a  force-
feed environment). The formulation should demonstrate acceptable toxicity; 

b)  test the palatability of the bait. The aim of the bait choice feeding trials is to determine 
the  palatability  of  the  product  for  the  test  insect.  In  this  test  design,  insects  have  the 
choice between a non-poisoned food source (challenge diet) and the bait containing the 
active substance; 

c)  the test should demonstrate acceptable toxicity in competition with the alternative food 

source; 

d)  assess  if  a  contaminated  group  of  termites  can  transfer  the  insecticide  to  a  group  of 
termites  that  have  never  been  exposed  to  it  before.  This  transfer  study  should 
demonstrate acceptable toxicity of termites not exposed directly to the baits. 

Laboratory/screening tests 

No-choice  test  (A):  test  the  termiticidal  efficacy  and  the  delayed  effect  of  an  insecticide 
formulation on a group of subterranean termites”: 

A group of termites is put into contact with an insecticide formulation. When testing baits, bait 
is  the  only  source  of  food.  For  other  types  of  termiticides  the  termites  are  exposed  to  the 
product  according  to  the  intended  use  (e.g.  spray  the  surface  and  add  the  termites  to  the 
surface. The test is performed in assay containers. Mortality of the insects is assessed. 

From  this  test  the  time  “te”  can  be  determined,  necessary  to  perform  the  test  B  (te=time  of 
exposure  of  the  termites  to  the  insecticide  formulation  which  is  required  to  observe  a 
significant mortality compared with termites in an untreated control).  

Transfer  test  (B):  the  transmission  of  the  insecticide  used  in  the  baiting  system  to  an 
uninfected group of termites: 

Termites  are  exposed  to  the  tested  bait  long  enough  to  be  contaminated  with  the  active 
substance (time te). A group of termites is removed from the colony and put in contact with a 
healthy  uncontaminated  group.  The  mortality  rate  of  both  groups  of  termites  (contaminated 
and uncontaminated) is assessed separately. 

Choice test / palatability test (C): the suppression of a group of termites reared in laboratory 
under conditions of food competition; with the use of the same insecticidal bait formulation: 

Add the insecticidal bait formulation to a group of termites already exploiting another source of 
food. The test is performed in assay containers. The aim is to assess the mortality after a given 
period of time. 

Field trial 

In field trials the product is tested in actual use situation and applied according to the direction 
for  use  on  the  label.  The  test  method  should  evaluate  the  efficacy  of  the  baits  or  barrier 
products in an experimental site where termite activity is reported. 

A common claim for a barrier product is the duration of “protection”. This is normally in terms 
of a number of years and should be demonstrated by long-duration soil tests in field plots. 

For  bait  products  consumption  of  the  tested  bait  must  be  registered  at  least  in  the  first  6 
months after the introduction of the baits. The elimination of termites in the experimental site 
should  be  registered  maximum  after  18  months  (counted  since  the  introduction  of  the  first 
tested bait), excluding the winter period. 

Table 27 gives an overview of available (French) guidelines for termites and how to use them. 

 
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Table 27: Overview guidelines on termites 

Preventive treatment/Physico-chemical barrier 

Protocol 

Ageing Test 

Laboratory test 

NF X 41-550 

after 
after 
after 

NF X 41-568 (effect of water) 
CTBA-BIO-E-016 (effect of the natural light) 
CTBA-BIO-E-007 (effect of alkalinity) 

Field test 

CTBA-BIO-E-008 

no 

Remedial treatment/chemical barrier 

Protocol 

Ageing Test 

Laboratory test 

NF X 41-550 

after 

NF X 41-542 (effect of water) 

Field test 
Wall chemical barrier 
Soil chemical barrier 

NF X 41-550 

after 

FCBA-BIO-E-053 

Remedial treatment/Bait system 

Protocol 

Ageing Test 

Laboratory test 

XP X 41-543-1  

Field test 

XP X 41-543-2 

no 

no 

5.6.4.5.3 Assessment of authorisation 

5.6.4.5.3.1 Norms and criteria 

A biocidal product may only be authorised if it “possesses a sufficient level of efficacy” (BPD). 
This is implemented in the following way. 

An  insecticidal  product  intended  for  the  control  of  termites  is  normally  considered  to  be 
sufficiently  “effective”  if  the  following  results  can  be  achieved  (derived  from  standards  NF 
XPX41-551, NF XPX41-543-3 and FCBA-BIO-E-041): 

Products intended for use as baits: 

•  no-choice  test:  100%  mortality  before  the  end  of  the  test  (16  weeks).  Besides,  if  the 
100%  mortality  is  achieved  too  fast  (less  than  48  hours)  the  test  bait  should  be 
rejected; 

• 

• 

transfer test: 100% mortality of all the termites, which have not been exposed directly 
with the tested bait; 

choice test / palatability test: more than 95% mortality; 

•  bait  field  test:  No  termite  activity  should  be  reported  within  the  test  period  (max.  18 
months, excluding the winter period). No termite activity should be reported in at least 
the following 3 months. 

Products intended for use as termite barriers 

• 

• 

laboratory test: 100% mortality after the test (only for barriers with lethal activity); 

field test: 

o 

o 

In soil barrier products, termites should not penetrate the soil more than 10 mm; 

In  wall  barriers  (i.e.  thermoplastic  films),  termites  should  not  be  able  to 
perforate the film after the duration of the test; 

 
 
 
 
 
 
 
 
 
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5.6.4.6 Bed Bugs 

5.6.4.6.1 Introduction 

Bedbugs are small, wingless blood feeding insects. Of the many recognized species, only three 
are known to feed on humans. In temperate climate regions of the EU, Cimex lectularius is the 
dominant species. Bedbugs are not known to transmit disease in Europe, but infestations  can 
cause painful and irritating bites on the skin while humans sleep. Once infested, treatment and 
control is very difficult. 

A  sign  of  bedbug  presence  include  bites  on  the  exposed  skin  (small  red  itchy  bumps)  of 
humans during sleep. If observed, confined locations such as mattress linings or furniture folds 
should be inspected for faecal spotting and the presence of bedbugs. 

5.6.4.6.1.1 Biology 

Bedbugs belong to the order of Hemiptera, Family Cimicidae. 

Bedbugs harbour themselves in very confined areas in wall cracks, furniture joints, along lining 
of  mattresses,  behind  pictures  and  in  seams  of  furnishings.  These  insects  generally  confine 
themselves to these areas and leave them only to feed. Bedbugs are negatively phototactic and 
not usually seen outside the harbourage in the day or when the lights are on. 

Female bedbugs can lay up to 500 eggs during their lifetime. Depending on frequency of blood 
meals,  bedbugs  can  live  for  more  than  a  year.  They  are  able  to  survive  for  months  without 
feeding (dependent upon temperature: at 16°C survival can be a year). The first nymph hatch 
from  small  white  eggs  after  7-10  days  at  room  temperature  (around  20°C)  and  earlier  at 
higher  temperatures.  Each  of  the  5  nymphal  stages  need  a  blood  meal  to  complete 
development  to  the  next  instar.  The  whole  life-cycle  from  egg  to  egg  takes  a  minimum  of  28 
days at 27°C or around 42 days at 22°C. 

5.6.4.6.2 Dossier requirements 

A clear label claim should be submitted. The study results of field trials should demonstrate the 
efficacy of the product, based on the submitted label claim. 

Laboratory  and  field  trials  with  bedbugs  are  needed  to  assess  the  efficacy  of  the  product. 
Ideally,  the  studies  should  be  performed  according  to  established  guidelines  where  these  are 
available. These may be international, EU or national guidelines. See Appendix  18 for a list of 
available guidelines. 

If there are no guidelines available or guidelines are not suitable, the applicant may use their 
own  methods,  on  condition  however,  that  the  study  is  scientifically  robust,  well  reported  and 
provides  a  clear  answer  to  the  question.  In  addition,  the  test  methods  applied  and  the  test 
conditions  should  be  clearly  and  fully  described  and  must  address  the  efficacy  claim  that 
appears on the product label. In the case of field trials where true replication is almost certainly 
impossible to achieve and where normal  control methods are not restricted to use of a single 
insecticidal  product,  a  full  description  of  any  factors  that  might  be  expected  to  influence 
product performance should be given. These may include the risk of re-invasion from adjacent 
areas,  general  levels  of  sanitation,  treatment  history  etc.  and  are  intended  to  provide  the 
authorities with information to assist with the interpretation of the results obtained. 

5.6.4.6.2.1 Test species 

A  product  against  bedbugs  should  normally  be  tested  on  the  common  bedbug  (Cimex 
lectularius) or tropical bedbug (Cimex hemipterus). 

5.6.4.6.2.2 Laboratory tests and field trials 

For  the  evaluation  of  biocides  against  bedbugs  different  types  of  laboratory,  simulated-use 
tests and field test can be used. Examples of tests are listed below. 

Screening studies (no- choice test) 

Testing  should  include  application  of  the  product  to  representative  surfaces  (e.g.  plywood, 
painted  plywood,  textile  fabric,  wallpaper)  or  direct  cuticle  application  of  the  product  to 
bedbugs  to  assess  inherent  contact  toxicity  of  the  active  substance.  It  should  be  specified 
whether  adults  or  nymphs  are  used.  A  test  may  be  used  to  demonstrate  basic  efficacy  or 
efficacy  against  insects  resistant  to  specific  chemicals  (LD50  versus  a  susceptible  field  or 

 
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laboratory  strain)  or  insect  growth  regulator  effects  (nymphs  are  treated  and  subsequent 
effects are recorded such as inhibition of moulting, deformities, sterile adults). 

Results  must  support  description  related  to  the  mode  of  action  (symptomology)  or  “effective 
against strains resistant to “x” class of insecticides”, or similar efficacy claims. 

Screening  tests  are  not  always  necessary.  It  is  sufficient  to  demonstrated  efficacy  in  residual 
tests or similar tests. 

Determination of residual efficacy 

Good  residual  efficacy  is  essential  for  insecticides  used  in  bedbug  control,  as  is  impossible  to 
treat all bedbugs directly or reach all of their hiding. 

For  the  determination  of  residual  efficacy,  the  formulated  product  (spray,  powder,  dust,  etc.) 
should be applied to representative surfaces at the recommended label rate. Bedbugs (adults) 
should be exposed to the deposit at several time intervals after the deposit has dried (including 
the day of treatment, but after the deposit has dried completely and at the end of the claimed 
residual  period).  Exposure  time  should,  preferably,  be  comparable  to  the  time  the  bedbugs 
might reasonably be expected to be in contact with a treated surface under practical conditions 
(e.g. 10 min - 6 hours) and assessors will take this  factor into consideration when  evaluating 
the  data.  Treated  surfaces  should  include  at  least  two  porous  and  one  non-porous  substrate, 
representing  surfaces  that  might,  typically,  be  treated  for  bedbug  control  (e.g.  plywood, 
painted  plywood,  textile  fabric,  wallpaper,  according  to  the  label  claim).  Mortality  is  normally 
assessed after 1 day up to 14 days post-exposure. 

For  insect  growth  regulators,  exposure  conditions  can  be  as  described  above,  but  selection  of 
the developmental stage (nymph, adult) and post-exposure assessment (deformities, moulting 
success,  sterility,  mortality)  must  be  adapted  to  suit  the  mode  of  action  of  the  active 
substance. Hence, assessments may continue to be made several weeks after exposure (sub-
lethal  or  non-lethal  effects  on  fertility,  sterility  for  example  may  contribute  to  long  term 
population control without short term mortality). 

Groups  of  bedbugs  should  be  of  specified  age/sex  and  number.  Tests  should  be  performed  in 
triplicate,  with  at  least  20  bedbugs  per  replicate.  When  5  or  more  replicates  are  used,  10 
insects per replicate are adequate. Replicates should preferably be conducted per applied dose, 
time point, and surface. Untreated surfaces must be included as negative controls. 

Environmental conditions must be specified for the test itself, and during storage of the treated 
substrates (temperature, humidity, photoperiod). Temperature would be expected to fall in the 
range  19-29°C.  For  use  in  Southern  European  countries  higher  temperatures  (up  to  40°C) 
might be necessary. 

A control treatment without biocide should be included in all laboratory trials. The control trial 
should  be  of  adequate  size  (i.e.  number  of  replications  and  individuals),  providing  sufficient 
statistical power and a fair impression of control mortality. 

Simulated use 

These tests are designed to mimic the practical use situation. The insects must have a choice 
to be in contact with the biocide or not. Due to the normal behaviour of the bedbugs, it seems 
to  be  very  difficult  to  design  simulated-use  tests  for  the  evaluation  of  products  for  bedbug 
control.  Bedbugs  do  not  leave  their  harbourage  during  daytime  and  without  a  host  which 
attracts them. 

Field trials 

In field trials the product is tested in actual use situations, for instance in an infested home or 
hotel and applied according to the direction for use on the label. 

It  has  to  be  considered  that  in  bedbug  infestations  the  aim  of  professional  control  operations 
must  be  the  eradication  of  the  population.  It  is  not  acceptable  to  have  even  very  small 
remaining  populations.  Usually,  pest  control  operations  against  bedbugs  have  to  combine 
different measures. The documentation of the trial has to give all information on the products 
or other measures used. 

5.6.4.6.2.3 Requirements per type of claim 

 
 
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Appropriate efficacy tests are needed for each claim. 

Products intended for use as general surface treatment for consumers: 

•  a  laboratory  test  showing  mortality  and/or  knockdown  and/or  residual  efficacy, 

depending on the claim. 

Products intended for use as general surface treatment for professionals: 

•  a  laboratory  test  showing  mortality  and/or  knockdown  and/or  residual  efficacy, 

depending on the claim; 

•  a simulated-use test showing mortality and knockdown according to the claim and/or; 

•  a field trial according to the directions for use; 

•  Simulated-use tests can be waived if a robust field trial is submitted. 

5.6.4.6.3 Assessment of authorisation 

5.6.4.6.3.1 Norms and criteria 

A biocidal product may only be authorised if it “possesses a sufficient level of efficacy” (BPD). 
This is implemented in the following way. 

An  insecticidal  product  intended  for  the  control  of  bedbugs  is  considered  to  be  sufficiently 
“effective” if the following results are achieved: 

Products intended for use as general surface treatment for consumers: 

• 

required results in laboratory tests (and simulated-use tests): 

o  ≥ 90% knockdown within a few minutes after contact with the product (or according 

to the claim), direct after application and at the end of the residual period; 

o  mortality according to the label claim, preferably ≥90% in 1 hour. 

Products intended for use as general surface treatment for professionals: 

• 

• 

required results in laboratory tests: 
o  direct application: 100% mortality within 24 hours after spraying the bedbugs; 
o  residual test: ≥ 95% mortality within 24 hours after placing the bedbugs in the test 

area, direct after spray and at the end of the residual period. 

required results in field test: 
o  after a period of 6-10 weeks, the population reduction exceeds 90% relative to either 

untreated sites or pre-treatment levels. 

Treatment  repeats  usually  are  necessary  in  bedbug  control.  At  the  end  of  a 
treatment, 100 % efficacy should be achieved. 

Deviations from these norms is possible but should be justified in the application. 

Data  from  field  trials  at  the  label  application  rate  must  preferably  be  evaluated  by  an 
experienced  assessor  since  performance  can  vary  considerably,  even  from  apartment  to 
apartment in the same building. The number of trials, the complexity of the trials sites, the use 
(or not)  of additional measures that can contribute to effective control, treatment history  etc. 
can  all  have  a  substantial  effect  upon  the  level  of  control  that  is  achieved.  The  data  must 
provide  evidence  of  suitable  levels  of  efficacy  during  the  residual  period  claimed,  relative  to 
pre-treatment population assessments and/or performance of reference products under similar 
conditions,  and/or  assessments  of  bedbug  populations  in  untreated  areas  under  similar 
conditions. Where mean population reduction exceeds 90% relative to either untreated sites or 
pre-treatment levels, the product is considered effective, but the assessor has the discretion to 
view each data set on its merits and consider all the factors before concluding whether the data 
support the claimed level of performance. 

5.6.4.7 Ticks 

5.6.4.7.1 Introduction 

Ticks are small arthropods classed along with mites and spiders in the Class Arachnida. All ticks 
are blood feeders. Certain tick species are known for carrying and transmitting many different 
pathogenic  micro-organisms  including  bacteria,  viruses,  parasites  and  fungi.  Diseases 
associated with tick transmission in Europe include Lyme disease, tick-borne encephalitis, and 
human  anaplasmosis,  all  transmitted  by  Ixodes  ricinus.  The  tick  Hyalomma  marginatum  can 

 
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transmit Crimean-Congo haemorrhagic fever, a viral disease common in East and West Africa. 
Mediterranean spotted fever is transmitted by the brown dog tick (Rhipicephalus sanguineus). 
Ticks also have an important role in animal health. They can cause anaemia, reduction of milk 
production and bodyweight gain of animals. 

5.6.4.7.1.1 Biology 

Ticks differ from insects morphologically having two main body parts (insects have three) and 
eight legs as nymphs and adults (six legs for insects). Ticks go through four stages to complete 
their lifecycle: egg, larva, nymph, and adult. Feeding will occur in both the immature and adult 
stages. After mating female hard ticks will feed once more followed by oviposition of hundreds 
to even thousands of eggs. 

Ticks can be differentiated on their host choices: 

•  one host: developing stages and adults feed on one host (e.g. Boophilus); 

• 

• 

two hosts: larvae and nymphs feed on the same host, adults feed on another host (e.g. 
Rhipicephalus); 

three  hosts:  larvae,  nymphs  and  adults  feed  on  three  different  hosts.  (e.g.  Ixodes, 
Haemophysalis, Dermacentor). 

Ticks can be classified into two main families: soft ticks (Argasidae) and hard ticks (Ixodidae). 
The  hard  ticks  consist  of  many  commonly  known  species  such  as  the  sheep  tick  (Ixodes 
ricinus),  the  brown  dog  tick  (R.  sanguineus)  and  Dermacentor  sp  .  H.  marginatum  is  also  a 
hard  tick.  Hard  ticks  vary  in  host-tick  relationship.  Species  may  have  one  host,  two  different 
hosts  or three different hosts. After mating female hard ticks will feed once more followed by 
oviposition of hundreds to even thousands of eggs. 

Soft ticks have similar body parts as the hard ticks. Key differences are that soft ticks lack the 
sclerotized outer cuticle found in hard ticks and the mouthparts of soft ticks are located below 
the  end  of  the  body  (hard  tick  mouthparts  stick  out  the  front  of  the  protected  hood).  For 
example the bird ticks, Argas reflexus and A. persicus, are soft ticks which can be a pest in for 
instance poultry farms. 

Hard ticks have to be fixed to their hosts and the meal can last five days, while soft ticks are 
not fixed and the meal is finished in 20 to 50 minutes. 

When  searching  for  a  possible  host,  ticks  generally  remain  stationary  until  a  host  passes  by. 
Once attached, ticks crawl to locate a place to feed. Commonly, ticks will attach to human skin 
along pant or sock lines or other tight locations which are warm and humid. Feeding can take 
hours to days depending on the species. 

The bird ticks, Argas persicus and A. reflexus have worldwide distribution in warm climates. A. 
persicus occurs in small poultry farms and feeds blood on chicken and other domestic fowls. A. 
reflexus  occurs  in  pigeon  farms  and  on  urban  pigeons  and  their  surroundings  in  towns.  They 
can  get  from  the  nests  of  pigeons  to  lofts  and  attic  rooms  and  feed  on  sleeping  humans  for 
blood. A. reflexus is an urban pest parasitizing urban pigeons and may cause a wide range of 
allergic reactions. 

Argas  spp.  hide  in  cracks  and  crevices  of  chicken  houses,  nests,  wooden  equipments  etc. 
during the day and come out to blood feed at night. Males and females are both blood sucking. 
They  are  able  to  survive  starvation  for  two  years,  which  is  why  the  protection  against  these 
mites is very difficult. 

5.6.4.7.2 Dossier requirements 

A clear label claim should be submitted. The study results of field trials should demonstrate the 
efficacy of the product, based on the submitted label claim. 

Laboratory and, for some claims, field trials with ticks are needed to assess the efficacy of the 
product.  The  studies  should  normally  be  performed  according  to  established  guidelines  where 
these are available. These may be international, EU or national guidelines. See Appendix 18 for 
a  list  of  available  guidelines.  If  no  guidelines  are  available,  the  applicant  may  use  their  own 
methods,  on  condition  however,  that  the  study  is  scientifically  robust,  well  reported  and 
provides  a  clear  answer  to  the  question.  In  addition,  the  test  methods  applied  and  the  test 
conditions  should  be  clearly  and  fully  described  and  must  address  the  efficacy  claim  that 
appears on the product label. In the case of field trials where true replication is almost certainly 

 
 
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impossible to achieve and where normal  control methods are not restricted to use of a single 
acaricidal product, a full description of any factors that might be expected to influence product 
performance  should  be  given.  These  may  include  the  risk  of  re-invasion  from  adjacent  areas, 
general levels of sanitation, treatment history etc. and are intended to provide the authorities 
with information to assist with the interpretation of the results obtained. 

5.6.4.7.2.1 Test species 

A  product  against  ticks  should  normally  be  tested  on  the  sheep  tick  Ixodes  ricinus.  When 
control  of  dog  or  bird  ticks  is  claimed,  tests  with  these  ticks  should  be  performed  too 
(Rhipicephalus  sanguineus,  A.  reflexus).  When  efficacy  in  the  tropics  is  claimed  or  efficacy 
against H. marginatum, this tick should be tested too. H. marginatum behaves differently than 
I. ricinus since it is aggressive and it actively seeks the host to feed on and moves quickely on 
the ground.  When the  product is intended for  use  in poultry farms tests  should be performed 
against A. persicus. 

5.6.4.7.2.2 Laboratory tests and field trials 

For  the  evaluation  of  biocides  against  ticks  different  types  of  laboratory  and  simulated-use 
tests can be used. Examples of tests are listed below. 

Laboratory test to evaluate knockdown and kill effect (no-choice test) 

A clear label claim should be submitted. The study results of field trials should demonstrate the 
efficacy of the product, based on the submitted label claim. 

Laboratory and, for some claims, field trials with ticks are needed to assess the efficacy of the 
product.  The  studies  should  normally  be  performed  according  to  established  guidelines  where 
these are available. These may be international, EU or national guidelines. See Appendix 18 for 
a  list  of  available  guidelines.  If  no  guidelines  are  available,  the  applicant  may  use  their  own 
methods,  on  condition  however,  that  the  study  is  scientifically  robust,  well  reported  and 
provides  a  clear  answer  to  the  question.  In  addition,  the  test  methods  applied  and  the  test 
conditions  should  be  clearly  and  fully  described  and  must  address  the  efficacy  claim  that 
appears on the product label. In the case of field trials where true replication is almost certainly 
impossible to achieve and where normal  control methods are not restricted to use of a single 
acaricidal product, a full description of any factors that might be expected to influence product 
performance  should  be  given.  These  may  include  the  risk  of  re-invasion  from  adjacent  areas, 
general levels of sanitation, treatment history etc. and are intended to provide the authorities 
with information to assist with the interpretation of the results obtained. 

Laboratory test for repellents 

Candidate repellents are applied to human forearms from wrist to elbow. As a negative control 
untreated arm will be tested, preferably the other arm of the test person. A line is drawn 3 cm 
above the wrist. Disease free ticks are placed  on the back  of the hand  with a forceps, 5 ticks 
per test. Fresh, starved ticks are  required for  each exposure.  The arm is inverted to promote 
upward  movement  toward  the  treated  surface  (ticks  are  negatively  geotropic).  The  first 
exposure is one hour post treatment and continues once an hour for four hours. Each exposure 
is  5  minutes.  Several  criteria  for  repellence  can  be  used.  Either,  a  tick  is  considered  non-
repelled if it crosses the line 3 cm above the wrist or a tick is considered repelled when it drops 
down  from  the  arm.  A  negative  control  treatment  on  an  untreated  arm,  preferably  the  other 
arm of the same test person, should be performed. 

Percentage  repellence  is  calculated  by  recording  the  number  of  ticks  crossing  the  line  or 
dropping down from a treated arm as opposed to a control arm. 

Normally,  per  repellent  at  least  10  persons  are  tested  since  repellence/attractiveness  to  ticks 
varies considerably between human individuals. 

An alternative method could be using animals instead of humans to test the repellence against 
ticks. I. ricinus, R. sanguineus and H. marginatum will bite both humans and animals. 

Simulated use tests 

To prevent disease transmission ticks must be knocked down, or killed before attaching to the 
skin. For products that knockdown and kill ticks a simulated-use tests should be performed in 
which  the  product  is  applied  according  to  the  instruction  for  use  and  then  tested  in  the 
presence of a person or an arm or foot or animal. For some products this can be a similar test 

 
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set  up  as  described  in  5.6.4.7.2.2.  Then  it  has  to  be  established  that  the  ticks  are  knocked 
down  or  killed  before  they  can  attach  to  the  skin  and  start  feeding.  This  is  compared  to  a 
control test. 

5.6.4.7.2.3 Requirements per type of claim 

• 

• 

Insecticide with knockdown or kill effect: laboratory and simulated-use tests. 

Simulated-use tests can be waived if a robust field trial is submitted. 

5.6.4.7.3 Assessment of authorisation 

5.6.4.7.3.1 Norms and criteria 

A biocidal product may only be authorised if it “possesses a sufficient level of efficacy” (BPD). 
This is implemented for ticks in the following way. 

An insecticide against ticks is normally  considered to be  sufficiently “effective” if the following 
results can be achieved: 

o   

•  Product with knockdown effect: 

o  100% knockdown before ticks start feeding and;  
o  ≥ 80% kill within 24 hours; 

•  Product with kill effect: 

o  ≥ 95% kill before ticks start feeding. 

Deviations from these norms is possible but should be justified in the application. 
5.6.4.8 Mites 

5.6.4.8.1 Introduction 

Mites,  along  with  ticks,  belong  to  the  subclass  Acarina  (also  known  as  Acari)  and  the  class 
Arachnida.  Mites  are  among  the  most  diverse  and  successful  of  all  the  invertebrate  groups. 
They have exploited an incredible array of habitats, and because of their small size (most are 
microscopic)  most  go  totally  unnoticed.  Perhaps  the  best-known  mite,  is  the  house  dust  mite 
(family  Pyroglyphidae),  which  can  cause  asthma  and  allergic  symptoms.  Mites  are  also 
important as vectors of micro-organisms, transmitting rickettsiae and  bartonellae. Flour mites 
(Acarus siro) and mould or storage mites (Tyrophagus putrescentiae, T. longior) are important 
pests  in  stored  goods.  Mites  like  the  red  mite,  Dermanyssus  gallinae,  can  be  a  pest  in  bird 
cages  and  poultry  farms.  The  red  mite  can  also  feed  on  some  species  of  mammals,  including 
humans, but need an avian host to reproduce. 

Part of the control of mites is covered in section 5.6.4.11 on stored goods. Often mites are only 
mentioned on a label as a secondary pest, while insects are the main pests. 

5.6.4.8.1.1 Biology 

The house dust mite is widespread in human habitation. House dust mites thrive in the indoor 
environment provided by homes, specifically in bedrooms and kitchens. Dust mites survive well 
in  mattresses,  carpets,  furniture  and  bedding,  with  figures  around  188  animals/g  dust.  Dust 
mites  feed  on  organic  detritus  such  as  flakes  of  shed  human  skin  and  flourish  in  the  stable 
environment  of  dwellings.  The  European  house  dust  mite  (Dermatophagoides  pteronyssinus) 
and  the  American  house  dust  mite  (Dermatophagoides  farinae)  are  two  different  species,  but 
are  not  necessarily  confined  to  Europe  or  North  America;  a  third  species  Euroglyphus  maynei 
also occurs widely. The average life cycle for a male house dust mite is 10 to 19 days. A mated 
female house dust mite can live for 70 days, laying 60 to 100 eggs in the last 5 weeks of her 
life. 

The  flour  mite,  A.  siro,  is  the  most  common  species  of  mite  in  foodstuffs.  The  males  are 
0.33 mm  to  0.43 mm  long  and  female  are  0.36  mm  to  0.66  mm  in  length.  Flour  mites 
contaminate  grain  and  flour  by  allergens  and  they  transfer  pathogenic  micro-organisms. 
Foodstuffs acquire a sickly sweet smell and an unpalatable taste. When fed infested foodstuff, 
animals show reduced feed intake, diarrhoea, inflammation of the small intestine and impaired 
growth. 

 
 
 
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The red mite, Dermanyssus gallinae, is an ectoparasite of poultry and birds. They can be found 
in  houses  of  laying  hens,  chickens  and  other  fowls.  The  mites  are  blood  feeders  and  attack 
resting birds at night. The optimal temperature is 27-28 °C. After feeding they hide in  cracks 
and crevices away from daylight, where they mate and lay about 30-35 eggs in their lifetime. 
Their maximal lifetime is 8 weeks without starving and 6-10 months with starving. In spite of 
that  these  mites  are  ectoparasites,  the  main  method  of  control  is  treating  of  the  walls,  bird 
cages, nests and hidden places in poultry farms with biocides. 

5.6.4.8.2 Dossier requirements 

A  clear  label  claim  should  be  submitted.  The  study  results  of  laboratory  or  field  trials  should 
demonstrate the efficacy of the product, based on the submitted label claim. 

 Laboratory and/or field trials with mites are needed to assess the efficacy of the product. The 
studies  should  normally  be  performed  according  to  established  guidelines  where  these  are 
available. These may be international, EU or national guidelines. See Appendix  18 for a list of 
available guidelines. If no guidelines are available or guidelines are not suitable, the applicant 
may use their own methods, on condition however, that the study is scientifically robust, well 
reported and provides a clear answer to the question. In addition, the test methods applied and 
the  test  conditions  should  be  clearly  and  fully  described  and  must  address  the  efficacy  claim 
that  appears  on  the  product  label.  In  the  case  of  field  trials  where  true  replication  is  almost 
certainly  impossible  to  achieve  and  where  normal  control  methods  might  not  be  restricted  to 
use  of  a  single  acaricidal  product,  a  full  description  of  any  factors  that  might  be  expected  to 
influence product performance should be given. These may include the risk of re-invasion from 
adjacent areas, general levels of sanitation, treatment history etc. and are intended to provide 
the authorities with information to assist with the interpretation of the results obtained. 

5.6.4.8.2.1 Test species 

Which test species should be used depends on the intended area of use and the label claim. In 
homes  the  European  house  dust  mites,  D.  pteronyssinus,  is  the  most  important.  In  storage 
rooms  the  flour  mite  or  storage  mites,  etc.,  for  instance  T.  putrescentiae,  A.  siro.  For  use  on 
poultry  farms  D.  gallinae  should  be  tested.  When  specific  mite  species  are  mentioned  in  the 
claim these should be tested. 

5.6.4.8.2.2 Laboratory tests and field trials 

For  the  evaluation  of  biocides  against  mites  different  types  of  laboratory  and  simulated-use 
tests can be used. Examples are given below. 

Laboratory test to evaluate knockdown and kill effect (no-choice test) 

The product is applied to representative surfaces or via direct cuticle application, in a container 
with mites, to assess inherent contact toxicity or knockdown effect of the active substance. For 
instance spray on a filter paper and put the filter paper in an aluminium dish. Specify whether 
adults  (male  or  female)  or  nymphs  are  used.  Normally  tests  are  performed  with  3  or  more 
replicates,  with  normally  20  to  30  mites  per  replicate.  Tests  are  done  at  25°C  and  70-75% 
R.H.. In all laboratory studies a treatment without biocide should be conducted with mites from 
the same population, as a negative control. The number of dead mites is counted at 24 hours 
after treatment. 

Residual effect 

For  determination  of  residual  efficacy,  the  formulated  product  should  be  applied  to 
representative  surfaces  at  a  specified  dose  rate,  or  rates,  including  the  recommended  label 
rate.  Mites  should  be  exposed  to  the  deposit  at  several  time  intervals  after  the  deposit  has 
dried (including the day of treatment, but after the deposit has dried completely and at the end 
of  the  claimed  residual  period).  Exposure  time  should,  preferably,  be  comparable  to  the  time 
the mites might reasonably be expected to be in contact with a treated surface under practical 
conditions  and  assessors  will  take  this  factor  into  consideration  when  evaluating  the  data. 
Treated  surfaces  should  include  at  least  two  porous  and  one  non-porous  substrate, 
representing  surfaces  that  might,  typically,  be  treated  for  mite  control  (e.g.  plywood,  painted 
plywood, textile fabric, according to the label claim). Mortality is normally assessed after 1 day 
up to 14 days post-exposure. 

Simulated use tests 

 
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These tests are designed to mimic the practical use situation. For products that knockdown and 
kill mites simulated-use tests should be performed in which the product is applied according to 
the instruction for use. When products for general surface treatment are tested the mites must 
have  a  choice  to  be  in  contact  with  the  biocide  or  not.  The  results  should  be  compared  to  a 
control test, without biocide. 

5.6.4.8.2.3 Requirements per type of claim 

Specific mites: when specific mite species are mentioned in the claim (e.g. dust mite, red mite) 
both laboratory and simulated-use tests are required with the target species. 

Mites  as  secondary  pest:  When  mites  are  mentioned  on  the  label  claim  only  as  a  secondary 
pest, only laboratory tests with one mite species are required. 

Acaricides:  When  mites  are  the  main  pest  to  control  both  laboratory  and  simulated-use  tests 
are required with more than one mite species.  

Space  and  structural  treatments:  requirements  for  these  products  are  covered  in  section 
5.6.4.11 on stored goods. 

Simulated-use tests can be waived if a robust field trial is submitted. 

5.6.4.8.3 Assessment of authorisation 

5.6.4.8.3.1Norms and criteria 

A biocidal product may only be authorised if it “possesses a sufficient level of efficacy” (BPD). 
This is implemented for mites in the following way. 

A  biocide  against  mites  is  normally  considered  to  be  sufficiently  “effective”  if  the  following 
results can be achieved: 

• 

• 

• 

laboratory tests: ≥90% mortality in 24 hours; 

simulated-use tests: ≥90%mortality in 1 week; 

field  trials  for  space  and  structural  treatments:  requirements  for  these  products  are 
covered in section 5.6.4.11 on stored goods. 

Deviations from these norms is possible but should be justified in the application. 
5.6.4.9 Fleas 

5.6.4.9.1 Introduction 

This section covers the assessment of  efficacy of products used for treatment against cat and 
dog fleas. The application of these products is indoors on surfaces. 

These  biocides  are  divided  into  two  groups,  namely  the  adulticidal  and  ovicidal/larvicidal 
products. Adulticidal products are intended for use against fleas in the adult growth stage, and 
the  ovicidal/larvicidal  products  for  use  against  fleas  in  the  egg  and  larval  stages.  This 
distinction  is  based  on  the  very  different  modes  of  action  of  the  product,  which  result  in 
different criteria for assessment. 

It  should  be  emphasized  that  products  against  fleas,  which  are  applied  directly  on  dogs  and 
cats  and  have  a  medical  claim  are  covered  by  legislation  on  Veterinary  Medical  Products.  The 
the  ECB  website 
to 
reader  may 
(www.ecb.jrc.it/biocides). 

the  borderline  dossier  available  on 

refer 

5.6.4.9.1.1 Biology 

Of  the  over  2000  species  of  fleas  (Siphonaptera),  the  cat  flea  (Ctenocephalides  felis)  and  the 
dog  flea  (C.  canis)  are  the  most  common  in-home  pests  in  the  EU.  Fleas  undergo  complete 
metamorphosis (egg, larva, pupa, adult) and the lifecycle begins when an adult female finds a 
suitable  host.  Once  found,  the  female  flea  will  remain  on  this  host  for  the  rest  of  its  life. 
Females produce several eggs after each blood feeding and can produce several hundred eggs 
in its lifetime. Once laid, the eggs fall off the animal host and develop in the areas where the 
host animal spends its time. The eggs tend to accumulate in the lowest areas such as deep in 
fibres of carpets, cracks in the floor, or crevices in furniture and furnishings. 

Larvae require high protein food for their survival. This protein comes from feeding on the dry 
faeces of the adult fleas. The adult flea takes in more blood from the host than necessary for 
nourishment and excretes the remaining blood in almost pure form. Once dried, the faeces falls 

 
 
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off  the  host  animal  where  the  larvae  can  feed.  The  larvae  spin  a  cocoon  and  begin  the  pupal 
state. 

An  adult  flea  emerges  from  the  pupae  after  stimulation  from  external  cues  that  indicate  an 
animal  host  is  near.  Once  emerged,  a  flea  must  usually  find  a  host  (located  using  visual  and 
thermal cues) within a week, or it risks death due to desiccation. Complete development from 
egg  to  adult  occurs  in  as  little  as  two  weeks,  but  this  can  take  much  longer  depending  on 
environmental conditions. 

5.6.4.9.2 Dossier requirements 

A  clear  label  claim  should  be  submitted.  The  study  results  of  laboratory.  simulated-use  tests 
and  field  trials  should  demonstrate  the  efficacy  of  the  product,  based  on  the  submitted  label 
claim. 

Laboratory and field trials with fleas are needed to assess the efficacy of the product. Ideally, 
the studies should be performed according to established guidelines where these are available. 
These  may  be  international,  EU  or  national  guidelines.  See  Appendix  18  for  a  list  of  available 
guidelines. If there are no guidelines available or the guidelines are not suitable, the applicant 
may use their own methods, on condition however, that the study is scientifically robust, well 
reported and provides a clear answer to the question. In addition, the test methods applied and 
the  test  conditions  should  be  clearly  and  fully  described  and  must  address  the  efficacy  claim 
that  appears  on  the  product  label.  In  the  case  of  field  trials  where  true  replication  is  almost 
certainly impossible to achieve and where normal control methods are not restricted to use of a 
single insecticidal product, a full description of any factors that might be expected to influence 
product performance should be given. These may include the risk of re-invasion from adjacent 
areas,  general  levels  of  sanitation,  treatment  history  etc.  and  are  intended  to  provide  the 
authorities with information to assist with the interpretation of the results obtained. 

5.6.4.9.2.1 Test species 

A product against fleas should normally be tested on the cat flea (Ctenocephalides felis) or the 
dog flea (C. canis). 

5.6.4.9.2.2  For  claims  made  for  products  intended  for  use  as  general  surface 
treatments 

For  the  evaluation  of  biocides  against  fleas  different  types  of  laboratory,  simulated-use  tests 
and field tests can be used. 

Examples  of  the  types  of  data  that  may  be  available  when  considering  the  efficacy  of 
insecticide products intended for use as surface treatments are given below. 

Laboratory studies 

The  product  is  applied  to  representative  surfaces  (e.g.  carpet  discs).  Information  on  the  fibre 
length  and  density  should  be  provided,  as  this  has  a  bearing  onto  flea  survival.  Long  fibres 
enable fleas to hide and, thus, protect fleas from getting their share of the insecticide applied, 
Fleas  are  transferred  to  the  surface,  either  before  (direct  contact)  or  after  (residual 
performance)  application  of  the  product,  to  assess  inherent  contact  toxicity  or  knockdown 
effect of the active substance. 

Alternatively, ovicidal or larvicidal products can be tested in flea rearing medium containing flea 
eggs  or  larvae  and  the  active  substance  in  a  range  of  concentrations,  including  the  intended 
use concentration. Preferably, tests should be done in five replicates per treatment. 

A control treatment without biocide with the  same number  of  replicates  should be included in 
all laboratory trials. 

Simulated use studies 

These  tests  are  designed  to  mimic  the  practical  use  situation.  The  test  should  be  performed 
according to the label claim. 

Simulated-use tests can be waived if a robust field trial is submitted. 

 
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5.6.4.9.2.3  For  claims  made  for  products  intended  to  be  used  as  space  spray 
treatments 

Some insecticides against fleas can be used in foggers. For the evaluation of these insecticides 
different types of laboratory, simulated-use tests and field tests can be used. 

The efficacy test design should be defined for the available treatment method. 

5.6.4.9.3 Assessment of authorisation 

5.6.4.9.3.1 Norms and criteria 

A biocidal product may only be authorised if it “possesses a sufficient level of efficacy”. This is 
normally implemented for fleas in the following way. 

For laboratory and simulated use: 

An adulticidal product against fleas is considered to be sufficiently “effective” if: 

•  within 24 hours 100% knockdown of the adult fleas should occur (this norm only applies 
if the test fleas are sprayed directly or are placed immediately on a treated carpet) and; 

•  within 48 hours ≥90% mortality of adult fleas should occur. 

An ovicidal/larvicidal product against fleas is considered to be sufficiently “effective” if: 

•  ≥80%  inhibition  should  occur  of  the  development  of  produced  eggs/larvae  into  adult 

fleas during the claimed ovicidal/larvicidal duration of action of the product. 

Deviations from these norms is possible but should be justified in the application. 

5.6.4.10 Litter Beetles 

5.6.4.10.1 Introduction 

There  are  several  species  of  "litter  beetles"  that  inhabit  poultry  droppings  and  litter.  Litter 
beetles  belong  to  the  order  Coleoptera,  family  Tenebrionidae.  The  most  important  are  the 
lesser mealworm (other names: darkling beetle), Alphitobius diaperinus, and two species in the 
dermestid  genus  Dermestes;  the  hide  beetle  (D.  maculatus)  and  the  larder  beetle  (D. 
lardarius).  Other  species  of  beetles  that  occasionally  cause  damage  to  poultry  housing  are 
Dermestes ater, Tenebrio mollitor, Alphitobius laevigatus, and Trox spp. 

Litter  beetles  are  of  particular  importance  as  a  vector  and  competent  reservoir  of  several 
poultry  pathogens  and  parasites.  The  transmission  of  bacteria,  (Salmonella,  Escherichia  coli) 
and  protozoa  (several  Eimeria  species  which  can  cause  coccidiosis)  and  different  viruses  can 
cause  problems  in  livestock.  This  pest  can  also  cause  damage  to  poultry  housing  and  is 
suspected  to  be  a  health  risk  to  humans  in  close  contact  with  larvae  and  adults.  Adults  can 
become a nuisance when they move en masse toward artificial lights generated by residences 
near fields where beetle-infested manure has been spread. 

Often these beetles are only mentioned on a label as a secondary pest, while other insects are 
the  main  pests  (control  of  flies,  cockroaches,  and  litter  beetles  in  poultry  houses).  But  when 
they are mentioned specifically on the label they should be tested. 

5.6.4.10.1.1 Biology 

Lesser mealworm adults lay their eggs in cracks and crevices in the poultry house, in manure 
or litter, and in grain hulls. Larvae hatch and complete development to the adult stage in 40-
100 days depending on temperature and food quality. The larvae consume spilled feed, manure 
and, to a lesser extent, dead birds and cracked eggs. Beetle populations in broiler and turkey 
houses  often are concentrated around lines of  feeders, which provide  the beetles with shelter 
and an opportunity to feed on spilled bird feed. Mature larvae disperse when they are crowded 
to  find  isolated  pupation  sites,  and  this  behaviour  is  responsible  for  much  of  their  destructive 
activity. Crowded larvae leave the litter and tunnel into thermal insulation materials where they 
construct  pupal  cells.  Both  larval  and  adult  stages  are  omnivorous.  The  lesser  mealworm  is 
nocturnal,  with  greatest  activity  of  both  larvae  and  adults  occurring  shortly  after  dark. 
Populations of lesser mealworm often reach high densities, especially in deep-litter broiler and 
turkey houses and in high-rise caged layer operations. It is not unusual for the litter of a broiler 
house to move from beetle activity or for 70% of the surface of manure in a high-rise house to 
be covered with adult beetles. 

 
 
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5.6.4.10.2 Dossier requirements 

A  clear  label  claim  should  be  submitted.  The  study  results  of  laboratory,  simulated-use  tests 
and  field  trials  should  demonstrate  the  efficacy  of  the  product,  based  on  the  submitted  label 
claim. 

Laboratory and simulated field trials with litter beetles are needed to assess the efficacy of the 
product.  Ideally,  the  studies  should  be  performed  according  to  established  guidelines  where 
these are available. These may be international, EU or national guidelines. See Appendix 18 for 
a  list  of  available  guidelines.  If  there  are  no  guidelines  available  or  the  guidelines  are  not 
suitable,  the  applicant  may  use  their  own  methods,  on  condition  however,  that  the  study  is 
scientifically robust, well reported and provides a clear answer to the question. In addition, the 
test  methods  applied  and  the  test  conditions  should  be  clearly  and  fully  described  and  must 
address  the  efficacy  claim  that  appears  on  the  product  label.  In  the  case  of  field  trials  where 
true replication is almost certainly impossible to achieve and where normal control methods are 
not restricted to use of a single insecticidal product, a full description of any factors that might 
be  expected to influence product performance  should be given. These may include the  risk  of 
re-invasion  from  adjacent  areas,  general  levels  of  sanitation,  treatment  history  etc.  and  are 
intended  to  provide  the  authorities  with  information  to  assist  with  the  interpretation  of  the 
results obtained. 

5.6.4.10.2.1 Test species 

A  product  against  litter  beetles  should  normally  be  tested  on  the  lesser  mealworm,  A. 
diapernus. 

5.6.4.10.2.2  For  claims  made  for  products  intended  for  use  as  general  surface 
treatments 

Examples  of  the  types  of  data  that  may  be  available  when  considering  the  efficacy  of 
insecticide products intended for use as surface treatments are given below. 

Laboratory studies 

The  product  is  applied  to  representative  surfaces,  either  before  (persistence  test)  or  after 
(direct contact) the insects are transferred to the surface, to assess inherent contact toxicity or 
knockdown effect of the active substance. 

Preferably, test should be done in five replicates per treatment. 

A control treatment without biocide should be included in all laboratory trials. 

Simulated use studies 

These  tests  are  designed  to  mimic  the  practical  use  situation.  The  test  should  be  performed 
according to the label claim. 

Simulated-use tests can be waived if a robust field trial is submitted. 

5.6.4.10.3 Assessment of authorisation 

5.6.4.10.3.1 Norms and criteria 

A biocidal product may only be authorised if it “possesses a sufficient level of efficacy”. This is 
normally implemented for litter beetles in the following way. 

A product against litter beetles is considered to be sufficiently “effective” if: 

For laboratory and simulated use: 

•  adulticide: ≥ 95% mortality; 

• 

• 

larvicide: ≥ 95% mortality; 

insect growth regulator: ≥ 90% mortality. 

Deviations from these norms is possible but should be justified in the application. 

 
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5.6.4.11 Textile-attacking Insects (including fur and fabric attaching insects) 

5.6.4.11.1 Introduction 

Insecticides  against  textile-attacking  insects  can  be  used  by  professionals  and  non-
professionals, use against beetle or moth larvae infested carpets for example.  

Home user products may be used in vapour phase to prevent moth contact with stored clothing 
(via  killing  moth  in  traps)  or  insecticides  may  be  applied  to  the  surface  of  clothing  to  kill 
landing moths on contact. 

Insecticides against textile-attacking insects can also be incorporated in the textile by industry 
for preventive treatments. 

Other  products  made  from  textiles  treated  with  insecticides  are  the  so-called  treated  articles 
with  an  external  claim  (e.g.  carpet  with  an  insecticide  not  to  protect  the  carpet  but  against 
fleas  that  are  in  contact  with  the  carpet).  These  treated  articles  will  not  be  considered 
specifically in this section since other than textile-attacking insects are the target insects. 

5.6.4.11.1.1 Biology 

The  two  main  orders  containing  textile  attacking  insect  species  are  Lepidoptera  (moths)  and 
Coleoptera  (beetles).  The  webbing  clothes  moth  (Tineola  bisselliella),  fur  moth  (Tinea 
pellionella), brown house moth (Hofmannophila pseudospretella) and carpet beetles (Anthrenus 
sp., Anthrenocerus sp.) are common in-house pests that feed on clothing, drapery, carpet and 
other  natural  hair  fibres.  The  larvae  of  these  insects  have  a  diet  consisting  of  natural  hair 
fibres, which provide protein from keratin in the hair. These insects have adapted to be able to 
digest keratin, which is not easily digested by other insects. 

Clothes  moths  are  distributed  worldwide.  They  feed  during  the  larval  cycle  within  a  silken 
cocoon attached to hair fibre. Clothes moths larvae that feed only on natural hair fibres such as 
wool, will not feed on, silk, cotton, linens or synthetic fibres. Adult clothes moths do not feed. 
These adults mate and the females lays eggs directly on the natural fibre food source. 

Carpet  beetle  larvae  (e.g.  Anthrenus  sp.,  Anthrenocerus  sp.)  attack  woollens,  rugs  and 
upholstered  furniture,  etc..  The  adult  beetles,  which  feed  on  nectar  and  pollen,  can  usually 
enter the home on plants, flowers or  other vegetation. Eggs are then laid on lint in protected 
areas such as behind baseboards. Once hatched, larvae begin feeding on a number of natural 
textiles or displays (animal horns, hoofs, insect collections, etc). 

5.6.4.11.2 Dossier requirements 

A clear label claim should be submitted. The study results of simulated-use tests or field trials 
should demonstrate the efficacy of the product, based on the submitted label claim. 

For  vapour  based  products  the  label  should  provide  information  on  the  volume  that  can  be 
covered with the product (closet of x m3, room of y m3). 

Laboratory  and  simulated-use  trials  with  textile-attacking  insects  are  normally  needed  to 
assess  the  efficacy  of  the  product.  Ideally,  the  studies  should  be  performed  according  to 
established  guidelines  where  these  are  available.  These  may  be  international,  EU  or  national 
guidelines.  See  Appendix  18  for  a  list  of  available  guidelines.  If  there  are  no  guidelines 
available  or  the  guidelines  are  not  suitable,  the  applicant  may  use  their  own  methods,  on 
condition  however,  that  the  study  is  scientifically  robust,  well  reported  and  provides  a  clear 
answer to the question. In addition, the test methods applied and the test conditions should be 
clearly  and  fully  described  and  must  address  the  efficacy  claim  that  appears  on  the  product 
label. In the case of field trials where true replication is almost certainly impossible to achieve 
and where normal control methods are not restricted to use of a single insecticidal product, a 
full description of any factors that might be expected to influence product performance should 
be  given.  These  may  include  the  risk  of  re-invasion  from  adjacent  areas,  general  levels  of 
sanitation, treatment history etc. and are intended to provide the authorities with information 
to assist with the interpretation of the results obtained. 

5.6.4.11.2.1 Test species 

A product against textile-attacking insects should normally be tested on: 

•  one of the following moth species: 

o  the clothes moth (Tineola bisselliella); 

 
 
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o  the fur moth (Tinea pellionella L.);  
o  the brown house moth (Hofmannophila pseudospretella ); 

•  one of the following carpet beetle species: 

o  Anthrenus sp;  
o  Anthrenocerus sp. 

Whether adults or larvae or both should be tested depends on the label claim. 

5.6.4.11.2.2 Laboratory tests and field trials 

For the evaluation of biocides against textile attacking insects different types of laboratory and 
simulated-use tests can be used. Examples of tests, mainly for cloths moth, are listed below.  

Laboratory tests 

Mortality test 
Webbing clothes moths, adults, larvae (2nd-3rd instar) or eggs may be placed in a jar (e.g. 240 
ml  glass  jars,  brass-screened  lid)  containing  a  treated  textile  sample  (e.g.  circular,  4cm 
diameter, 100% wool sample). 

Jars  are  periodically  evaluated  by  recording  mortality,  egg  laying  and  hatch  (optional),  and 
larval  damage.  A  moth  is  considered  inactivated  when  it  is  not  able  to  walk  or  fly,  in  a 
spontaneous way or when stimulated with a brush or pin. 

New moths are introduced into the jars periodically to test  residual  effects (depending  on the 
label  claims).  Tests  should  normally  be  done  in  five  replicates.  A  control  treatment  without 
biocide with the same number of replicates should be included in all laboratory trials. 

Simulated use 

These tests are designed to mimic the practical use situation. The study results should provide 
a clear picture of the efficacy of the product. 

An example of tests that might match the proposed intended use of the product: 

Simulated-use tests with moths added to drawers (minimum air volume: 0.016 m3) or closets 
(minimum air volume: 0.5 m3) can provide good information on home user products. In tests 
with  vapour  based  products  the  door  should  be  opened  with  a  frequency  resembling  normal 
opening of a closet, to show that this does not reduce efficacy: once a day during completion of 
the assay, 5 seconds for drawers and 10 seconds for  closets. Assessments of mortality would 
form  the  basis  for  efficacy  claims.  Additionally  damage  to  the  test  material  can  be  assessed. 
The damage will depend upon the number of insects, their developmental stage, the exposure 
time and the size and quality of the piece of carpet, etc. Therefore, damage should always be 
assessed in comparison to the control treatment. 

Simulated-use tests can be waived if a robust field trial is submitted. 

Test  similar  to  the  ones  mentioned  above  can  also  be  used  to  show  efficacy  against  carpet 
beetles and the larvae of carpet beetles. 

5.6.4.11.3 Assessment of authorisation 

5.6.4.11.3.1 Norms and criteria 

A biocidal product may only be authorised if it “possesses a sufficient level of efficacy”. This is 
implemented for textile attacking-insects normally in the following way. 

At the end of an exposure period (e.g. 1 week): 

•  more than 90% of the adults and larvae should be killed (unless claimed different); 

Deviations from these norms is possible but should be justified in the application. 
5.6.4.12 Stored Goods-attacking Insects and Mites 

5.6.4.12.1 Introduction 

The purpose of biocidal products against stored goods-attacking insects and mites is to control 
pests  in  storerooms,  freight  and  alternative  transport  containers  for  products  of  plant  origin 
etc.  They  should  also  protect  the  actual  stored  goods  against  insects  and  mites.  The  term 
“stored”  in  this  regard  refers  specifically  to:  stored  products  (of  plant  origin)  for  human 
consumption, animal feed, industrial processing and propagation. 

 
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Products  against  stored  goods-attacking  insects  can  either  be  biocides  or  plant  protection 
products. In general, where the stored products are protected, prior to processing, the use falls 
under plant protection and is not relevant in this guideline. 

There  are  a  number  of  different  insects  that  attack  stored  goods.  Common  beetle  invaders 
include  grain  beetles  (Tribolium  castaneum,  Oryzaephilus  surinamensis,  etc.),  confused  flour 
beetles  (Tribolium  confusum),  and  rice  weevils  (Sitophilus  oryzae).  Indian  meal  moth  (Plodia 
interpunctella)  and  flour  mite  (Acarus  siro)  are  also  very  common  pest.  Infestations  of  these 
pests  can  occur  at  the  packaging  plant,  the  store,  or  in  the  home,  making  it  difficult  to 
determine  where  the  source  of  the  problem  is.  Sometimes  these  infestations  are  only  noticed 
by the consumer once the insect leaves the food product and enters the home environment. 

For professional and industrial use there are two classifications of such products: 

• 

fumigation with gases, which is used for controlling pests in rooms used for the storage 
of  products  of  plant  origin  (storerooms,  freight  structures  and  means  of  transport, 
gassing installations etc.); 

•  products  other  than  gases,  which  are  used  for  controlling  pests  in  empty  or  full 

storerooms (including products which are applied by means of vaporisers). 

5.6.4.12.2 Dossier requirements 

A clear label claim should be submitted. The study results of simulated-use tests and field trials 
should demonstrate the efficacy of the product, based on the submitted label claim. 

Laboratory and field trials with stored goods-attacking insects are needed to assess the efficacy 
of  the  product.  Ideally,  the  studies  should  be  performed  according  to  established  guidelines 
where these are available. These may be international, EU or national guidelines. See Appendix 
18 for a list of available guidelines. EPPO standards PP 201 to 204 are recommended (Appendix 
18).  If  these  guidelines  are  not  suitable,  the  applicant  may  use  their  own  methods,  on 
condition  however,  that  the  study  is  scientifically  robust,  well  reported  and  provides  a  clear 
answer to the question. In addition, the test methods applied and the test conditions should be 
clearly  and  fully  described  and  must  address  the  efficacy  claim  that  appears  on  the  product 
label. 

In  the  case  of  field  trials  where  true  replication  is  almost  certainly  impossible  to  achieve  and 
where  normal  control  methods  are  not  restricted  to  use  of  a  single  insecticidal  product,  a  full 
description  of any factors that might be expected to influence product performance should be 
given.  These  may  include  the  risk  of  re-invasion  from  adjacent  areas,  general  levels  of 
sanitation, treatment history etc. and are intended to provide the authorities with information 
to assist with the interpretation of the results obtained. 

5.6.4.12.2.1 Test species 

A  product  against  stored  goods-attacking  insects  may  be  tested  on  beetles,  moths  or  mites 
(more  specifically  mentioned  in  the  relevant  EPPO  guidelines),  or  insects  that  are  specifically 
identified in the label claim. 

5.6.4.12.2.2 Laboratory tests and field trials 

Depending  on  the  application  and  the  purpose  of  the  product,  one  of  the  trials  below  (or 
equivalent trials) normally should be performed. 

Consumer products 

For  consumer  products  laboratory  or  simulated-use  tests  are  required.  A  direct  spray  test 
method  can  be  used  to  evaluate  performance  against  stored  goods-attacking  insects.  A 
simulated use test  can  be a test, performed in a laboratory, where insects (either cultured or 
natural populations) are in contact with the stored goods (e.g. breakfast cereal, flour) and the 
biocide is applied according to the instructions for use. 

Simulated-use tests can be waived if a robust field trial is submitted. 

A control treatment without biocide with the  same number  of  replicates  should be included in 
all laboratory trials. 

Gases  for  use  in  storerooms,  freight  and  transport  rooms  and  gassing  installations 
with stored products present 

 
 
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Additional laboratory studies are not required, only field trials. 

A  field  trial  should  normally  be  conducted  according  to  the  EPPO  guideline  PP  1/201(1) 
“Fumigants to control insect and mite pests of stored plant products”. 

The  field  of  use  of  the  gas  are  places  where  large  supplies  are  stored,  in  particular  cereal 
products,  but  also  other  food  products  such  as  dried  nuts,  processed  vegetables,  spices  or 
meals. 

The  use  of  gas  can  be  intended  for  controlling/fighting  pests  in  spaces  but  also  for 
controlling/fighting pests in or on the product itself. 

Products other than gases for storerooms with or without stored products 

Additional laboratory studies are not required, only field trials. 

A field trial normally should be performed according to the EPPO guideline PP 1/202 (1) “Space 
and structural treatments of storerooms”. 

The  products  concerned  exclude  gases,  but  do  include  those  applied  by  means  of  vaporisers 
(fogs, smokes, vapours, space sprays). 

This trial focuses on the control of pests in full or empty storerooms (walls, cracks, etc.). The 
trial  does  not  serve  to  test  the  efficacy  of  the  treatment  on  pests  in  the  stored  products 
themselves. 

The trial can be performed in two ways. 

• 

• 

the first possibility is conducting the trial in rooms where there is already an infestation. 
Using  a  trapping  system,  the  effectiveness  is  determined  by  scoring  the  number  of 
insects caught in the traps before and after the treatment; 

the second possibility is conducting the trial in a room where test organisms have been 
introduced  artificially  (usually  in  small  cages).  The  effectiveness  is  determined  by 
scoring the number of alive, ‘knocked down’ and dead organisms in comparison with an 
untreated room. 

5.6.4.12.3 Assessment of authorisation 

5.6.4.12.3.1 Norms and criteria 

A biocidal product may only be authorised if it “possesses a sufficient level of efficacy”. This is 
implemented for stored goods attacking-insect in the following way. 

• 

consumer  products:  normally  100%  mortality  in  direct  spray  tests,  in  simulated-use 
tests >90% knockdown and >70% mortality after 24 hours would be sufficient; 

•  gases:  the  duration  of  gassing  (as  specified  in  the  label  claim)  should  be  such  that  at 
the  end  of  gassing  100%  of 
insects/mites  are  dead  or  dying. 
the 
It  is  possible  to  distinguish  between  dead  and  dying  insects,  which  will  not  recover 
anymore, so these should also be counted; 

• 

the duration of gassing should not be longer than necessary; 

•  all  non-gases:  the  effect  should  be  achieved  within  the  duration  of  the  treatment,  as 

specified in the label claim. Normally >90% would be sufficient. 

Deviations from these norms is possible but should be justified in the application. 

5.6.4.13 Flies 

5.6.4.13.1 Introduction 

Flies are common pests in and around the house and in animal rearing facilities. Some of these 
insect species are merely a nuisance, others provide discomfort from irritating bites, and some 
potentially carry and transmit diseases. 

The  possible  fields  of  use  of  the  insecticides  include:  residential  and  other  types  of 
accommodation, public  spaces, hospitals, storerooms, kitchens,  waste dumps and stables and 
manure storage facilities. 

5.6.4.13.1.1 Biology 

House flies (Musca domestica) and other nuisance flies are common non-biting pests in the EU. 
The house fly lifecycle goes through four stages: egg, larvae (maggots), pupa, and adult. Eggs 

 
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are laid on organic debris including faeces, decaying vegetation, etc. Once hatched, larvae feed 
by burrowing into the organic debris and filter decaying organic matter. In the pupal stage the 
fly  is  transformed  into  the  adult.  During  this  transformation,  no  feeding  takes  place.  At  the 
adult stage, house flies feed by regurgitating on food, then lap up the food in liquid form. The 
life cycle of house flies, from egg to fly, is 1 to 3 weeks, depending on the climate conditions. 
Males  die  soon  after  mating,  females  live  temperature  dependent  normally  one  to  several 
weeks in the field. 

Flies  regularly  fly  into  and  out  of  man-made  structures.  Outside,  flies  land  on  faecal  material 
and  other  debris.  Inside,  flies  land  on  human  food  and  contact  other  substrates  regularly 
touched by humans. Here, potential pathogens  can be transferred on the flies’ body (legs) or 
from inside the body (vomiting on potential food in order to feed) which are picked up in faecal 
or other decaying material. More than 100 germs have been documented as being transferred 
by  house  flies.  Among  them  are  Salmonella  sp.  and  E.  coli  have  been  documented  as  being 
transferred by house flies. 

The stable fly (Stomoxys calcitrans) is a pest often found in stables alone or together with the 
housefly.  Rather  unusual  for  a  member  of  the  family  Muscidae  is  that  it  sucks  blood  from 
mammals. Under favourable conditions the stable flies develop from egg to fly in 3 weeks. The 
adults live several weeks. 

Other  biting  flies  include  black  fly  (Simuliidae)  and  deer  and  horse  flies  (Chrysops  and 
Tabanids), are also common pests in the EU. These insects can inflict a painful bite leaving an 
itchy  welt.  Some  are  also  known  to  transmit  disease.  Apart  from  these  species  blow-flies  can 
be of significance in a number of localities, including food producing facilities (Carrion flies, blue 
bottle fly, green bottle flies). 

5.6.4.13.2 Dossier requirements 

A clear label claim should be submitted. The study results of laboratory and simulated-use tests 
and  field  trials  should  demonstrate  the  efficacy  of  the  product  based  on  the  submitted  label 
claim. 

Laboratory, simulated-use tests and field trials  with the test insects are needed to assess the 
efficacy of the product, depending on the label claim. Ideally, the studies should be performed 
according to established guidelines where these are available. These may be international, EU 
or  national  guidelines.  See  Appendix  18  for  a  list  of  available  guidelines.  If  there  are  no 
guidelines  available  or  the  guidelines  are  not  suitable,  the  applicant  may  use  their  own 
methods  (intra-company  Standard  Operating  Procedures),  on  condition  however,  that  the 
study  is  scientifically  robust,  well  reported  and  provides  a  clear  answer  to  the  question.  In 
addition, the test methods applied and the test conditions should be clearly and fully described 
and  must  address  the  efficacy  claim  that  appears  on  the  product  label.  A  control  treatment 
without biocide with the same number of replicates should be included in all laboratory trials. 

In  the  case  of  field  trials  where  true  replication  is  almost  certainly  impossible  to  achieve  and 
where  normal  control  methods  are  not  restricted  to  use  of  a  single  insecticidal  product,  a  full 
description  of any factors that might be expected  to influence product performance should be 
given.  These  may  include  the  risk  of  re-invasion  from  adjacent  areas,  general  levels  of 
sanitation,  treatment  history,  season,  etc.  and  are  intended  to  provide  the  authorities  with 
information to assist with the interpretation of the results obtained. 

5.6.4.13.2.1 Test species 

In  case  of  an  authorisation  against  flies  the  prescribed  test  insect  is  the  housefly  (M. 
domestica).  When  the  product  claim  includes  use  in  stables  and  animal  housings  (except 
poultry),  for  a  general  claim  against  flies  both  the  housefly  and  the  stable  fly  (S.  calcitrans) 
should be tested. If efficacy against blow-flies is claimed tests have to be done with a blow-fly 
species (Calliphoridae).  

5.6.4.13.2.2 Laboratory testing simulated use tests and field trials 

For  evaluation  of  biocides  against  flies  different  types  of  laboratory,  simulated-use  tests  and 
field test can be used. Examples of tests are listed below. 

Laboratory tests 

 
 
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Flies can be tested in the laboratory in small jars or Petri dishes. The surface can be treated or 
granules  can  be  placed,  after  which  insects  can  be  added  at  different  time  intervals. 
Alternatively, the flies  can be sprayed directly. The  knockdown percentages and mortality are 
determined. 

A control treatment without biocide with the  same number  of  replicates  should be included in 
all laboratory trials. 

Simulated use tests 

For  assessment  of  efficacy  simulated-use  tests  should  be  conducted  in  a  test  chamber,  for 
instance the Peet-Grady chamber. This is an airtight room of 1.8 x 1.8 x 1.8 m3, into which a 
certain  amount  of  product  is  introduced.  Other  chambers  of  similar  or  bigger  size  are 
acceptable,  either  airtight  or  with  air  exchanges.  The  chamber  should  be  washed  and  dried 
between each replicate to avoid chemical contamination. 

Environmental  conditions  must  be  specified  during  the  test  (temperature,  humidity, 
photoperiod).  Temperature  would  be  expected  to  fall  in  the  range  19-29°C,  may  be  lower  for 
use in stables. A control treatment without biocide with the same number of replicates should 
be included in all laboratory trials. 

Simulated-use tests can be waived if a robust field trial is submitted. 

Examples  of  tests  for  different  products  are  listed  below.  For  other  types  of  products  similar 
test can be performed. 

Space treatment 
In  the  case  of  an  application  of  a  liquid  for  space  treatment,  the  aerosol  test  method  is 
performed  in  the  test  chamber  in  the  laboratory.  A  known  number  (50-100)  test  insects, 
including  males  and  females,  are  exposed  to  the  space  treatment.  The  dose  sprayed  in  the 
chamber should be comparable to the label directions. The test is performed in quadruplicate. 
A  control  treatment  without  biocide  should  be  included.  The  knockdown  percentages  and 
mortality of flies in both insecticide treatment and negative control are determined. 

Surface treatment 
Products for surface treatment (including window stickers) act on the insect via contact with or 
feeding from the treated surface. The product  can be applied by spraying, brushing, painting, 
etc. according to the label. These products are also tested in the test chamber. 

In  the  test  chamber  the  product  is  applied  on  a  small  surface  or  on  the  whole  chamber,  in  a 
dose rate appropriate to the label claim. After the surfaces have been left to dry the test can 
commence. The insects are released in the test chamber at several time points after application 
(or at least at the maximum residual time claimed at the label), to show residual efficacy. At a 
suitable  period  of  exposure  (e.g.  24  hours)  after  each  test  time  point  mortality  of  the  test 
insects  is  recorded.  It  is  mandatory  to  report  temperature  and  air  humidity  in  the  test  room. 
These should agree as much as possible with practical use conditions. 

Products to be vaporized or fogged 
Only a French recognized guideline (NF T 72-321) is available for efficacy studies with products 
against flies that should be vaporized (heating element that heats a tablet or liquid, coils, fan 
driven devices, etc.) or products that should be applied in a fogging treatment. Recently WHO 
published a guideline  for these types of products against mosquitoes.  This guideline might be 
adapted for fly products. Further, the “Large room test” is generally accepted. Other methods 
are also acceptable if they are scientifically sound and provide a clear picture of the efficacy of 
the product. 

The “Large room test” test can be performed in a non-ventilated room of 20 to 30 m3. When a 
ventilated  room  is  used  (mimics  in  some  cases  reality  better)  the  air  exchange  should  be 
measured (e.g. one air chamber renovation per hour). The product is applied according to the 
intended  use,  allowing  it  to  evaporate  over  a  specified  time  period  (depending  on  the  label 
claim e.g. 9 hours). 

House flies (M. domestica) are exposed to the vapour/fog at different time points, e.g. at 0, 2, 
4,  6  and  8  hours.  The  test  insect  to  be  used  depends  on  the  requested  application.  At  every 
time point a known number of test insects (e.g. 50), including males and females, are exposed 
to  the  vapour.  The  test  is  performed  in  quadruplicate.  A  control  treatment  without  biocide 
should be included. 

 
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The  knockdown  percentages  (KD50,  KD95,  KD100),  mortality  and, 
concentration of the active substance in the room are determined. 

if  possible,  the 

Larvicides 
Larvicides are often applied to the floor of stables and to manure to prevent maggots and pupa 
from  developing  into  the  next  stage.  These  products  can  be  tested  in  naturally  or  artificially 
infested  manure,  in  boxes  covered  with  gauze.  Adult  flies  emerging  from  the  manure  are 
counted  and  the  difference  between  treated  and  untreated  manure  is  analysed.  Where  IGRs 
(insect  growth  regulators)  are  used  as  larvicides,  it  is  possible  to  additionally  assess  the 
deformation of larvae and pupae. 

Bait products 
For  products  formulated  as  baits  the  product  should  also  be  tested  to  establish  the  intrinsic 
palatability of the formulation. 

The most important factor involved in laboratory testing is to provide a free choice alternative 
food  source  to  the  test  insects.  The  formulation  should  demonstrate  acceptable  toxicity  in 
competition with the alternative food source. A control treatment without biocide of similar size 
as the test itself (i.e. number of replications) should be included in all laboratory trials. 

If conducted on both fresh and aged product it may provide information on the storage stability 
of the product. 

Field trials 

For  application  in  cattle  houses,  pigsties  and/or  treatment  of  pig  and  cattle  manure  for 
controlling flies, field trials are normally required. 

Tests are done preferably during spring and beginning of summer. At the end of summer and 
autumn population decline might be due to natural causes instead of the insecticide treatment. 
Apply the insecticide according to the label instructions. 

During field trials in stables, special consideration should be given to the choice of the building 
material  (concrete,  wood  etc.)  of  the  walls  and  floors  of  the  stables,  as  well  as  to  the 
ventilation  (number  of  total  air  changes  per  24  hours),  because  the  conditions  should  be 
representative  of  a  practical  situation.  This  can  differ  per  EU  country.  It  is  possible  to  assess 
whether  extrapolation  to  other  types  of  accommodation  is  justified.  If  for  example  a  general 
registration  for  poultry  houses  is  requested,  but  studies  conducted  in  a  house  for  laying  hens 
have been submitted, a rational should be provided that extrapolation is justified. 

The effect on the fly population can be determined by counting the numbers of flies (estimation 
of  population  size)  before,  during  and  after  the  treatment,  or  by  the  differences  between 
treated  and  untreated  objects  in  the  same  area.  Various  assessment  methods  are  acceptable 
including visual assessments (fly density on a surface or animals is assigned to a category) or 
quantified  measures  such  as  using  sticky  fly  papers,  digital  photographs  of  marked  areas  on 
walls, collecting dead flies from a defined floor or aisles area etc. 

5.6.4.13.2.3 Requirements per type of claim 

Per type of claim the requirements will be listed. 

Products  intended  for  use  as  general  surface  treatment,  space  treatment  or  vaporisers  in 
houses: 

•  a  simulated-use  test  showing  mortality  and  knockdown  and/or  residual  efficacy 

according to the claim. 

Products  intended  for  use  as  general  surface  treatment,  space  treatment  or  vaporisers  in 
stables and waste dumps: 

•  a  laboratory  test  showing  mortality  and/or  knockdown  and/or  residual  efficacy, 

depending on the claim; 

•  a field trial according to the directions for use. 

Products intended for use as larvicides:  

•  a laboratory test showing larva mortality; 

•  a simulated-use test showing decrease in number of emerging flies. 

Simulated-use tests can be waived if a robust field trial is submitted. 

 
 
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5.6.4.13.3 Assessment of authorisation 

5.6.4.13.3.1 Norms and criteria 

A  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient  level  of  efficacy”.  An 
insecticide against flies is considered to be sufficiently “effective” if the following results can be 
achieved: 

Products  intended  for  use  as  general  surface  treatment,  space  treatment  or  vaporisers  in 
houses:  

• 

required results in simulated-use tests: 

o  the level of knockdown efficacy should be ≥80%; 
o  mortality after 24 hour should be >90%. 

Products  intended  for  use  as  general  surface  treatment,  space  treatment  or  vaporisers  in 
stables and waste dumps:  

• 

• 

required results in laboratory tests: 

o  the level of knockdown efficacy should be ≥80%; 
o  mortality after 24 hour should be ≥90%; 

required results in field trials: 

o  reduction  in  the  amount  of  flies  according  to  the  claim  (or  compared  to  the  control 

situation). 

Products intended for use as larvicides: 

• 

required results in laboratory tests: 
o  >90% larva mortality; 
o  showing decrease in number of emerging flies. 

Deviations from these norms is possible but should be justified in the application. 

5.6.4.14 Mosquitoes 

5.6.4.14.1 Introduction 

Mosquitoes, including species in the Culex, Aedes, and Anopheles Genera are common pests in 
parts  of  the  EU.  As  well  as  their  annoying  behaviour  and  itching  bites,  mosquitoes  are  well-
known for transmitting diseases such as Malaria (Anopheles spp.), yellow fever, Dengue (Aedes 
spp.),  West  Nile  (e.g.  Culex  spp.),  blue  tongue  virus  in  animals,  and  various  encephalitis. 
Although  none  of  these  diseases  are  endemic  in  Europe,  occasional  outbreaks  occur  and 
European  travellers  might  encounter  them,  either  in  European  tropical  overseas  regions  or  in 
the  rest  of  the  world.  Biocides  against  mosquitoes  can  only  claim  to  kill  or  repel  the 
mosquitoes, not to prevent the diseases. 

5.6.4.14.1.1 Biology 

Like  all  Diptera,  mosquitoes  also  go  through  four  stages  of  development.  The  egg,  larval  and 
pupal  stages  take  place  in  still  aquatic  environments  such  as  floodplains,  drainage  ditches, 
natural  and  artificial  water  containers.  Depending  on  the  species,  female  mosquitoes  will  lay 
eggs  directly  in  these  aquatic  environments  or  adjacent  to  locations  in  mud  which  typically 
have fresh water or tidal flooding events. Depending on the genera,  eggs are laid individually 
or in clumps called rafts. 

Once  larvae  hatch,  filter  feeding  begins  near  the  top  of  the  water.  Typically,  mosquitoes  go 
through  4  larval  instars  before  beginning  the  pupal  stage.  Once  completed,  mosquito  adults 
emerge from the aquatic and enter the aerial environments. Mating usually begins a few hours 
to days after emergence. Once mated, the females begin to search for a blood meal. Humans 
and  domestic  animals  are  included  as  potential  blood  hosts,  with  some  mosquito  species 
preferring human blood to other animals. 

Adult  female  mosquitoes  locate  potential  blood  hosts  by  detecting  attractants  such  as  carbon 
dioxide and skin emanations. Once located, the mosquito will attempt to bite, taking in a blood 
meal. This blood meal is partially digested and used for the development of eggs. 

 
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5.6.4.14.2 Dossier requirements 

A  clear  label  claim  should  be  submitted.  The  study  results  of  trials  should  demonstrate  the 
efficacy of the product based on the submitted label claim. 

Laboratory, simulated-use tests and field trials  with the test insects are needed to assess the 
efficacy  of  the  product.  Ideally,  the  studies  should  be  performed  according  to  established 
guidelines  where  these  are  available.  These  may  be  international,  EU  or  national  guidelines. 
See Appendix 18 for a list of available guidelines. Several WHO tests are available for mosquito 
testing.  If  the  available  guidelines  are  not  suitable,  the  applicant  may  use  their  own  methods 
(intra-company  Standard  Operating  Procedures),  on  condition  however,  that  the  study  is 
scientifically robust, well reported and provides a clear answer to the question. In addition, the 
test  methods  applied  and  the  test  conditions  should  be  clearly  and  fully  described  and  must 
address  the  efficacy  claim  that  appears  on  the  product  label.  A  control  treatment  without 
biocide should be included in all laboratory trials. 

In  the  case  of  field  trials  where  true  replication  is  almost  certainly  impossible  to  achieve  and 
where  normal  control  methods  are  not  restricted  to  use  of  a  single  insecticidal  product,  a  full 
description  of any factors that might be expected to influence product performance should be 
given.  These  may  include  the  risk  of  re-invasion  from  adjacent  areas,  general  levels  of 
sanitation,  treatment  history,  season,  etc.  and  are  intended  to  provide  the  authorities  with 
information to assist with the interpretation of the results obtained. 

5.6.4.14.2.1 Test species 

In case of an authorisation against mosquitoes insecticide testing should be performed with the 
house  mosquito  (Culex  spp.)  since  this  is  the  most  common  in  Europe  and  a  large  mosquito, 
which makes it one of the most difficult to kill.  

When  use  in  tropical  areas  is  claimed  it  should  be  specified  against  which  mosquito  spp.  the 
product is effective and these should be tested (e.g. malarial mosquitoes: Anopheles). 

5.6.4.14.2.2 Laboratory studies 

For  the  evaluation  of  biocides  against  mosquitoes  different  types  of  laboratory,  simulated-use 
tests and field test can be used. Examples of test are listed below. Mosquitoes used in all tests 
should be disease free. 

Laboratory tests against adults 

Insecticides  against  mosquitoes  should  normally  be  tested  in  the  laboratory  in  WHO  cones  or 
WHO  cylinders  by  force  tarsal  contact.  The  test  is  well  described  in  WHO  guidelines 
(methodology, number, age, nutritional status of the specimens and insecticide susceptibility of 
the strains). Only females have to be tested. First laboratory test (bio assay) can be conducted 
on a laboratory strain of well-known insecticide susceptibility. A second test can be conducted 
on field populations obtained by larval collection. Tests should be conducted on F1 generation 
adults. Mosquitoes are  exposed during a few  minutes to a treated surface and their  evolution 
(knock  down,  death)  is  followed  during  24  hours.  The  knockdown  percentages  and  mortality 
are determined. 

The cone tests can also be used to evaluate the efficacy of insecticide treated net. For netting 
evaluation the exposure time is only 3 minutes and mortality is also checked after 24 hours. 

Tunnel  tests  baited  with  birds  or  little  mammals  could  be  conducted  to  assess  the  feeding 
inhibition and the insecticide effect. 

A control treatment without biocide with an adequate number of replicates should be included 
in all laboratory trials. 

Laboratory tests: Larvicides 

Larvicides  are  applied  to  water  to  prevent  larva  to  develop  into  adult  mosquitoes.  These 
products can be tested  in naturally or artificially infested  water, in boxes covered with gauze. 
Tests  are  normally  not  performed  in  tap  water  but  in  water  containing  organic  particles, 
especially where a claim for residual performance is made. Test is normally performed on late 
3rd-early  4th  larval  stages  only.  Mortality  is  usually  checked  after  24  hours.  For  slow  acting 
insecticides and insect growth regulators mortality has to be checked for several days. In that 
case  food  has  to  be  supplied  to  larval  stages.  A  control  population  susceptible  to  insecticide 

 
 
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should be use as control in all bio-assays (positive control). A control treatment without biocide 
should be included as negative control. Adult mosquitoes emerging from the water are counted 
and  the  differences  between  treated  and  untreated  boxes  are  analysed.  The  methodology  of 
this bio-assay is described in WHO guidelines (WHO/CDS/WHOPES/GCDPP/2005.13). 

Simulated use tests 

For  assessment  of  efficacy  simulated-use  tests  should  be  conducted  in  a  test  chamber,  for 
instance  the  Peet-Grady  chamber.  This  is  an  airtight  room  of  1.8*1.8*1.8  m,  into  which  a 
certain  amount  of  product  is  introduced.  Other  chambers  of  similar  or  bigger  size  are 
acceptable. 

The  chamber  should  be  washed  and  dried  between  each  replicate  to  avoid  chemical 
contamination. 

Next to chambers experimental huts can be used. These huts are small buildings, several build 
next  to  each  other,  in  which  wild  mosquitoes  can  enter  but  they  have  no  way  to  escape. 
Volunteers are in the huts as attractants for mosquitoes. In each hut, the treatment of the hut 
(space  or  surface  treatment)  or  the  volunteers  (skin  repellents)  should  be  different:  test 
product,  negative  control  (no  biocide)  or  positive  control  (standard  product  e.g.  DEET  for 
repellents). At the end of a test period (e.g. one night) number of mosquitoes are counted by 
species, by status (death or alive), by  engorgement (fed  or unfed) and by position in the hut 
(hut  or  exit  traps).  Advantage  of  the  hut  is  that  wild  populations  can  be  used  and  that  it  is 
ventilated (mimics reality better in some cases). 

Environmental conditions must be specified at the beginning and during the test (temperature, 
humidity,  photoperiod).  Temperature  would  be  expected  to  fall  in  the  range  19-29°C.  When 
efficacy at high temperatures is claimed (use in the tropics) test at temperatures >30°C should 
be provided. A control treatment without biocide should be included in all laboratory trials. 

Simulated-use tests can be waived if a robust field trial is submitted. 

Space treatment simulated use tests 
In  the  case  of  an  application  for  a  liquid  for  space  treatment,  the  aerosol  test  method  is 
performed  in  the  test  chamber  in  the  laboratory.  A  known  number  (e.g.  50-100)  test  insects 
(females)  are  exposed  to  the  space  treatment.  The  dose  sprayed  in  the  chamber  should  be 
comparable  to  the  label  directions.  The  test  is  replicated  3  or  more  times.  The  knockdown 
percentages and mortality of mosquitoes in both insecticide treatment and negative control are 
determined. Ideally, a ventilated room should be used to mimic the intended use better. 

 Surface treatment simulated use tests 
Products for surface treatment act on the insect by tarsal contact with the treated surface. The 
product  can  be  applied  by  spraying,  brushing,  painting,  etc.  according  to  the  label.  These 
products  are  also  tested  in  a  test  chamber  or  an  experimental  hut.  The  WHO  guideline  for 
testing mosquito adulticides describes such a test. 

In  the  test  chamber  the  product  is  applied  on  small  surface,  or  on  the  whole  chamber,  in  a 
dose  rate  appropriate  to  the  label  claim.  A  negative  control  should  be  included.  After  the 
surfaces  have  been  left  to  dry  the  test  can  commence.  The  insects  are  released  in  the  test 
chamber  at  several  time  points  after  application  (or  at  least  at  the  maximum  residual  time 
claimed at the label), to  show residual  efficacy. After 24 hours mortality of the test insects is 
recorded.  It  is  mandatory  to  report  temperature  and  air  humidity  in  the  test  room.  These 
should agree as much as possible with practical use conditions. 

Products to be vaporized or fogged simulated use tests 
No officially recognized guidelines are available for efficacy studies with products that should be 
vaporized  (heating  element  that  heats  a  tablet  of  liquid,  coils,  fan  driven  dives,  etc.)  or 
products  that  should  be  applied  in  a  fogging  treatment.  The  “Large  room  test”  is  generally 
accepted. Other methods are also acceptable if they are scientifically sound and provide a clear 
picture of the efficacy of the product. 

The “Large room test” test can be performed in a non-ventilated room of 20 to 60 m3. When a 
ventilated  room  is  used  (mimics  reality  better  in  some  cases)  the  air  exchange  should  be 
measured (e.g. one air chamber renovation per hour). The product is applied according to the 
intended  use,  allowing  it  to  evaporate  over  a  specified  time  period  (depending  on  the  label 
claim e.g. 9 hours). 

 
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Mosquitoes  are  exposed  to  the  vapour/fog  at  different  time  points,  e.g.  at  0,  2,  4,  6  and  8 
hours. At every time point a known number of female test insects (50-100) are exposed to the 
vapour. The test is replicated 3 or more times. A negative control should be included. 

The  knockdown  percentages  (KD50,  KD95,  KD100),  mortality  and, 
concentration of the active substance in the room are determined. 

if  possible,  the 

When the label claim says that the product should be used in ventilated rooms the opening of 
windows and doors should be simulated in the test. 

Repellents 
Repellents are products with a repellent effect and can drive away mosquitoes. These products 
are either applied on human or animal skin or on clothes (topical or skin repellent) or release 
the active ingredient to the air (spatial repellents).This is based on the biological activity of the 
evaporated active substance. 

Products with a repellent effect, which are applied on the human skin or clothes, can be tested 
in  an  “arm-in-cage”  simulated-use  test.  The  repellent  is  applied  in  the  specified  dose  on  the 
(bare) forearm of the test person. The forearm is subsequently exposed to test mosquitoes in a 
cage  for  5  minutes.  This  should  be  repeated  every  hour,  at  least  up  to  the  claimed  efficacy 
period. If one bite is received during an exposure followed by another bite in the next exposure 
(confirming the first bite), the test should be stopped and the time  of the first confirmed bite 
recorded  as  the  length  of  repellence.  If  bites  are  not  received  in  succession,  then  the  test  is 
continued and the first bite should be considered ‘unconfirmed’. The same test is repeated with 
untreated  forearms  of,  preferably,  the  same  test  persons.  For  the  untreated  forearm,  a 
minimum of 5 lands in 5 minutes is required to qualify the test. Once 5 lands are received, the 
arm should be removed to prevent excess biting. If less than 5 lands are counted in 5 minutes, 
then  the  test  should  not  proceed  and  the  mosquito  cage  should  be  replaced  with  ‘fresh’ 
mosquitoes. The results of treated and untreated forearms are compared. 

Alternative  methods  using  rabbits  are  developed.  The  repellent  solution  could  be  applied 
directly on the skin of a rabbit on which a cage containing female mosquitoes is placed. 

Skin  repellents  can  also  be  tested  in  and  experimental  hut,  as  long  as  the  number  of 
mosquitoes entering the hut is not too low. 

Similar tests can be used for cloth in which a repellent is incorporated (treated article). 

Repellent  effectiveness  is  based  on  protection  time,  that  is,  the  time  between  repellent 
application  and  the  time  of  2  or  more  bites  on  the  treated  arm,  or  the  first  confirmed  bite  (a 
bite followed by another within 30 min.). 

For products with a repellent effect, which are applied in another way (not on the human skin 
or clothes, for instance spatial repellents), no common protocols are available. These products 
can be tested in a simulated use test, for instance in an experimental hut. The submitted data 
from  studies  are  checked  for  completeness,  based  on  the  applied  dose  per  treated  area.  It  is 
also  checked  whether  the  duration  of  exposure  is  sufficient.  If  the  formulation  alone  i.e. 
without  the  carrier  (e.g.  a  product  with  a  tissue  as  carrier)  has  been  tested,  data  on  release 
from the carrier are also required. The study data should provide a clear picture of the efficacy 
of the product. 

When the label claim says that the product should be used in ventilated rooms the opening of 
windows and doors should be simulated in the test. 

Larvicides simulated use tests 
In  small  scale  simulated-use  tests,  insecticide  formulation  can  be  tested  in  natural  breeding 
sites or simulated larval breeding sites. When natural larval populations are used pre-treatment 
assessments  of  the  population  should  done  at  the  site  (larval  count  by  dipping  technique). 
Depending  on  the  protocol,  eggs  or  larvae  can  be  regularly  introduced  in  the  treated  sites  to 
evaluate  the  residual  efficacy.  Breeding  sites  are  kept  uncovered  to  allow  wild  adults  to  lay 
their  eggs.  The  methodology  of 
in  WHO  guidelines 
(WHO/CDS/WHOPES/GCDPP/2005.13). 

is  described 

test 

this 

Field trials 

For  some  products  against  mosquitoes,  field  trials  are  not  required.  Especially  when  field 
populations  are  used  in  the  lab  or  in  an  experimental  hut.  However,  for  some  products  and 

 
 
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uses a simulated-use test cannot mimic the practical situation sufficiently (e.g. larvicides used 
in large swamps and lakes, aerial applications). Especially with aerial applications the way the 
product  is  dispersed  can  make  a  difference  for  efficacy.  In  these  cases  the  competent 
authorities should require a field test. 

Tests  are  done  preferably  during  spring  and  beginning  of  summer.  In  autumn  population 
decline might be due to natural causes instead of the insecticide. Larvicides should normally be 
tested in July-August when sufficient levels of Culex spp. and Aedes spp. can be found. In any 
field  trial,  the  assessment  of  efficacy  requires  pre-  and  post-treatment  assessments  of  the 
population. CDC light traps are one commonly used method to trap mosquitoes and can provide 
both  quantitative  (how  many  mosquitoes)  and  qualitative  (which  species  are  present)  data. 
Other  methods  (exhauster,  aspirator)  can  be  used  too.  Apply  the  insecticide  according  to  the 
label instructions. 

5.6.4.14.2.3 Requirements per type of claim 

Per type of claim the requirements will be listed. 

Products  intended  for  use  as  general  surface  treatment,  space  treatment  or  vaporisers  in 
houses: 

•  a laboratory test showing adult mortality; 

•  a  simulated-use  test  showing  mortality  and  knockdown  and/or  residual  efficacy 

according to the claim. 

Products intended for use as larvicides: 

•  a laboratory test showing larva mortality; 

•  a simulated-use test showing decrease in number of emerging mosquitoes; 

•  depending on the  claim (mandatory for use in  natural waters)  field test showing larval 

mortality or decrease in number of emerging mosquitoes. 

Products intended for use as repellent on skin or clothes:  

•  a simulated-use test (arm-in-cage) showing repellence; 

•  a field study showing repellence in the field. 

Products intended for use as repellent not on skin or clothes:  

•  a laboratory and/or simulated-use test showing repellence; 

•  depending on the claim field test showing repellence. 

Products intended for use as repellent on horses (recreational and/or sport horses): 

• 

• 

a laboratory test demonstrating repellence; 

a  simulated  use/  field  test  demonstrating  repellence  against  the  specific  target 
mosquito species on target animals. 

Simulated-use tests can be waived if a robust field trial is submitted. 

5.6.4.14.3 Assessment of authorisation 

5.6.4.14.3.1 Norms and criteria 

A  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient  level  of  efficacy”.  An 
insecticide against mosquitoes is considered to be sufficiently “effective” if the following results 
can be achieved: 

Products  intended  for  use  as  general  surface  treatment,  space  treatment  or  vaporisers  in 
houses: 

• 

required results in simulated-use tests: 

the level of knockdown efficacy should be >80%; 

o 
o  mortality after 24 hour should be >90%. 

Products intended for use as larvicides:  

• 

required results in laboratory tests: 

o  100%  mortality  after  24  hours  of  contact  is  usually  required.  For  slow  acting 
insecticide  100%  mortality  after  48,  72  hours  or  more  could  be  considered. 
Exceptionally  a  larval  mortality  >90%  can  be  acceptable  if  all  the  surviving 
larvae died before or during emergence; 

 
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• 

required results in simulated-use or field tests: 
o  >90% larva mortality; 
o  showing decrease in number (usually 80%) of emerging mosquitoes. 

Deviations from these norms is possible but should be justified in the application. 

5.6.4.15 Wasps 

5.6.4.15.1 Introduction 

There  are  two  types  of  wasp  control:  control  of  the  wasps’  nest  and  control  of  single  flying 
wasps entering a home. The control of wasps’ nests may be performed both indoors (in cavity 
walls or attics), as well as outdoors (in trees, under roof gutters). 

5.6.4.15.1.1 Biology 

The  major  pest  wasps  (Hymenoptera)  are  the  social  wasps  in  the  family  Vespidae.  Yellow-
jackets  ((Para)Vespula  spp.,  Dolichovespula  spp.),  paper  wasps  (Polistes  spp.),  and  hornets 
(Vespa spp.) all belong to this family and are the greatest pests to homeowners. Wasps can be 
easily differentiated from bees by the fact that a wasp’s body appears to be hairless and their 
hind legs thinner than a bees. 

The vespid or social wasp lives in colonies in nests built of a paper-like material. Each nest is 
begun in the spring by a single queen who has mated the previous autumn. The queen builds a 
small  nest  in  which  she  begins  to  lay  eggs.  It  is  only  non-fertile  female  worker  wasps  that 
emerge from these initial eggs. These workers take over the nest building duties and forage for 
food  to  feed  the  larvae  that  emerge  from  subsequent  eggs.  Some  of  these  eggs  are  fertile 
females and some are males. 

Mature  colonies  are  divided  into  a  social  order  consisting  of  the  queen,  workers,  males,  and 
fertile females. In the autumn, the males and newly produced queens leave the nest to mate. 
The  male’s  sole  purpose  is  to  inseminate  the  fertile  females,  which  will  become  next  year’s 
queens.  The  newly  inseminated  queens  will  then  find  a  sheltered  place  where  they  will 
hibernate to begin the cycle with building a new nest the following spring. 

Unprovoked,  wasps  are  not  aggressive  stingers  but  will  protect  themselves  and  their  nests 
making them an undesirable occupant of properties and buildings. Wasps commonly infiltrate in 
and  around  homes  in  search  of  nest  sites  and  areas  to  hibernate  causing  problems  for  the 
homeowner.  Some  people  are  allergic  to  wasp  venom,  and  can  have  life-threatening  allergic 
reactions. Unlike bees, wasps can sting repeatedly. 

For effective control of wasps, the entire wasps’ nest should be treated. The control is aimed at 
exterminating all wasps that are within the nest that can fly. If this is achieved, the eggs and 
larvae  that  are  still  present  cannot  be  taken  care  of  and  fed  anymore,  resulting  in  the 
elimination of the entire nest. 

5.6.4.15.2 Dossier requirements 

A clear label claim should be submitted. The study results of field trials should demonstrate the 
efficacy of the product based on the submitted label claim. 

Laboratory  and  field  trials  with  the  test  insects  are  needed  to  assess  the  efficacy  of  the 
product.  Ideally,  the  studies  should  be  performed  according  to  established  guidelines  where 
these are available. These may be international, EU or national guidelines. See Appendix 18 for 
a  list  of  available  guidelines.  If  there  are  no  guidelines  available  or  the  guidelines  are  not 
suitable,  the  applicant  may  use  their  own  methods  (intra-company  Standard  Operating 
Procedures),  on  condition  however,  that  the  study  is  scientifically  robust,  well  reported  and 
provides  a  clear  answer  to  the  question.  In  addition,  the  test  methods  applied  and  the  test 
conditions  should  be  clearly  and  fully  described  and  must  address  the  efficacy  claim  that 
appears  on  the  product  label.  A  control  treatment  without  biocide  should  be  included  in  all 
laboratory trials. 

In  the  case  of  field  trials  where  true  replication  is  almost  certainly  impossible  to  achieve  and 
where  normal  control  methods  are  not  restricted  to  use  of  a  single  insecticidal  product,  a  full 
description  of any factors that might be expected to influence product performance should be 
given.  These  are  intended  to  provide  the  authorities  with  information  to  assist  with  the 
interpretation of the results obtained. 

 
 
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5.6.4.15.2.1 Test species 

A product for use against wasps should be tested on colonies and/or workers of Vespula spp. or 
Dolichovespula spp. 

5.6.4.15.2.2 Laboratory simulated use tests and field studies 

For the evaluation of biocides against wasps different types of laboratory and field test can be 
used. Examples of test are listed below. 

Laboratory tests 

Wasps can be tested in the laboratory in small jars or Petri dishes. The individual wasps should 
have sufficient access to food (e.g. sugar solution), since they can starve to death within hours 
when isolated from their nest without food. The surface can be treated, after which insects can 
be  added  at  different  time  intervals.  Alternatively,  the  wasps  can  be  sprayed  directly. 
Concentrations used must be in accordance with the claim. The knockdown percentages and/or 
mortality and/or residual effect are determined. 

A control treatment without biocide with a similar number of replications should be included in 
all laboratory trials. 

Field trials 

Insecticides with a claim to kill wasps’ nests should be tested in a field trial. The efficacy of the 
product  should  be  tested  in  at  least  5  nests.  Depending  on  the  label  claim  different  nests 
(locations) should be tested (e.g. free hanging in trees or on buildings, hidden in the soil or in 
wall  cavities,  etc.).  A  few  like  size  nests  should  be  monitored  over  the  same  test  period  as 
untreated controls. A pre-treatment activity count should be taken over a pre-determined time 
interval  of  both  treated  and  untreated  nests.  A  well-established  parameter  for  wasp  colony 
activity  is  the  traffic  rate,  which  is  defined  as  the  number  of  wasps  entering  and  leaving  the 
colony in a given time. The traffic rate can be determined 7 days before the treatment for at 
least 5 minutes at two different times of day as well as on the day of  treatment in order get a 
picture  of  the  colony  activity  and  development.  The  time  interval  between  both  observations 
must be at least 2 h. Treatment should be consistent with label instructions. When the nest is 
visible  it  can  be  treated  directly.  In  some  cases  the  nest  is  hidden,  for  instance  in  between 
walls  or  ceiling  of  houses.  In  those  cases  normally  all  the  openings  through  which  the  wasps 
enter  the  space  in  which  the  nest  is  hidden  should  be  treated.  Nest  position,  number  of 
entrances as well as wasp species must be described. 

After 24 hours, one week and two weeks post-treatment the activity or lack thereof should be 
recorded  by  determination  of  the  traffic  rate  at  the  treated  and  untreated  nests.  The  check 
after  one  and  two  weeks  is  required  since  it  is  possible  that,  when  pupae  are  not  eliminated, 
wasps emerging from pupae can take over the duties of feeding the larvae. 

5.6.4.15.2.3 Requirements per type of claim 

Products intended for the control of the wasps’ nest: 

• 

field trial with at least 5 treated nests. 

Products intended for the control of flying wasps: 

• 

laboratory or simulated-use test. 

5.6.4.15.3 Assessment of authorisation 

5.6.4.15.3.1 Norms and criteria 

A  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient  level  of  efficacy”.  For 
wasps this is implemented in the following way. 

Products intended for the control of the wasps’ nest: 

• 

required results in a field test: 
o  in 80% of the treated nests mortality of the flying wasps should be 100% within 24 
hours and all of the treated nests must have 100% mortality (i.e. no visible signs of 
nest activity) after one and two weeks. 

Products intended for the control of flying wasps: 

• 

required results in a laboratory or simulated-use test: 
o  ≥  90%  knockdown  within  a  5  -10  minutes  after  contact  with  the  product  (or 

 
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according to the claim), direct after spray and at the end of the residual period; 

o  mortality according to the label claim, preferably 90% in 1 hour. 

5.6.5 PT19 Repellents and attractants 

5.6.5.1 Introduction  

This  chapter  covers  repellents  and  attractants  under  the  BPR  which  are  not  regulated 
otherwise,  e.g.  plant  protection  products,  medical  devices,  medicinal  human  and  veterinary 
products. Depending on its field of use a product to repel or attract organisms, e.g. arthropods, 
molluscs,  leeches,  snakes,  mammals,  birds,  etc.  may  be  classified  as  a  biocidal  product. 
Monitoring traps are not in the scope of this guidance. 

Efficacy  evaluation  for  attractants  in  PT18  bait  products  should  be  done  on  the  basis  of  the 
requirements  for  bait  products  (see  PT18  Chapter:  Insecticides,  acaricides  and  products  to 
control other arthropods). 

The requirements for efficacy evaluation for PT19 products (except for products against stored 
goods-attacking  insects  and  mites)  do  not  distinguish  between  products  for  professional  and 
non-professional use. 

This  first  section  gives  a  general  introduction  and  lists  requirements  that  are  common  to  all 
sections. The following sections describe the specific requirements for efficacy testing for each 
target organism. 

Information is missing on some arthropods, molluscs, leeches, snakes, mammals, birds, etc. to 
be  repelled  or  attracted  and  also  on  some  of  the  uses  and  types  of  products.  In  such  cases, 
applicants  should  design  a  test  protocol  and  contact  their  prospective  CA  to  agree  upon  it. 
These data gaps will be filled in a future update of this guidance. Nevertheless, efficacy should 
be demonstrated according to general principles described in this guidance, notably simulated-
use or field tests should be performed. 

5.6.5.1.1 Aim 

The aim of this document is to provide guidance on how to assess the efficacy of repellents and 
attractants,  in  order  to  ensure  that  only  sufficiently  effective  products  are  authorised  and 
therefore placed on the market. Animal welfare considerations are also taken into account. 

5.6.5.1.2 Global structure of the assessment  

A  full  assessment  of  efficacy  is  required  for  applications  for  product  authorisations.  For  active 
substance approval test protocols described in the relevant sections can be used.  

Factors  to  be  considered  during  efficacy  assessment  for  PT19  biocidal  products  are  the 
following (non-exhaustive list): 

• 

• 

• 

• 

the target organism (including developmental stage and sex, if relevant) to be repelled 
or attracted; 

the formulation of the product (e.g. liquid/powder/bait/gas); 

the application method and rate; 

the frequency of treatment and any specific interval between applications;  

•  other  specific  conditions  (use  of  the  product  in  conjunction  with  other  activities:  e.g. 
cleaning  of  an  area  prior  to  treatment;  contributions  made  by  other  components  of  an 
Integrated Pest Management plan); 

• 

the areas of use, e.g.: 

- 

- 

- 

repellents intended for use as topical repellents for human or animal skin; 

repellents applied on clothing and on other treated articles; 

repellents  applied  on  surfaces  (indoor  and/or  outdoor  environments),  spatial 
repellents (indoor and/or outdoor environments); 

-  attractants  in  traps  without  PT18  active  substances  and  dispensers  (mating 

disruption). 

 
 
 
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Information on the effectiveness and intended uses of the product must be sufficient to permit 
an evaluation of the product and to define its conditions of use. 

Efficacy  tests  (see  relevant  sections  for  required  test  details)  should  be  performed  with  the 
product  such  as  marketed  for  which  the  authorisation  is  sought.  The  studies  should 
demonstrate  that  the  product  in  the  final  product  configuration  (co-formulant(s),  packaging  if 
relevant  and  active  substance(s))  is  effective  for  the  intended  use(s)  at  the  specified  dose(s) 
and use conditions (please note special requirements for Biocidal Product Families, see section 
5.6.5.1.3.2).  The  name  and  CAS/EC  number(s)  of  the  active  substance(s)  and  its 
percentage(s)  used  in  the  test  must  be  provided  in  the  efficacy  reports.  The  assessment  will 
consider  the  target  organism(s),  indoor  or  outdoor  use,  the  method(s)  of  application, 
application  rates  and  use  patterns  of  the  product,  maximum  storage  period  of  the  product, 
together  with  any  other  specific  claims  made  for  the  product.  The  use(s)  specified  in  the 
application,  i.e.  in  the  draft  Summary  of  Product  Characteristics  (SPC),  submitted  by  the 
applicant represent(s) the basis for evaluation. 

5.6.5.1.3 Dossier requirements 

The following guidance is designed to be flexible and does not specify rigid protocols to which 
tests  must  be  conducted.  Published  or  unpublished  data  relevant  to  the  application  will  be 
considered  if  valid.  In  all  cases,  the  methods  and  results  have  to  be  described  in  sufficient 
detail to make the data reproducible and to allow a full assessment. Anecdotal evidence will not 
be acceptable. 

The studies should normally be performed according to established guidelines where these are 
available. These may be international, EU or national guidelines (ISO, CEN, OECD, WHO, etc.). 
See Appendix 18 for a list of available standard test methods. 

If no guidelines are available or the guidelines are not suitable, the applicant may use elements 
of  their  own  methods  (intra-company  Standard  Operating  Procedures,  test  protocols  or  study 
plans), on the condition however, that the study plan and report are scientifically robust, well 
reported and provide clear scientifically based results. The test methods and the test conditions 
must clearly and fully be described and must address the efficacy claim appearing in the SPC. 

The  following  information  on  effectiveness  is  required  for  each  biocidal  product  in  accordance 
with Annex III of the BPR: 

•  function and mode of action (including time delay, residual efficacy, shelf-life); 

•  representative  target  organism(s)  (appropriate  to  the  geographic  regions  the  product 

will be used); 

•  organisms or objects to be protected; 

•  effects on relevant target organism(s); 

•  documentations of undesirable or unintended side effects, for example on beneficial and 

other non-target organism(s) in case of field trials; 

•  names of active substances and their respective concentration in the tested formulation 
and, if needed, e.g. aged product testing, batch number and date of manufacture of the 
product; 

•  statement  about  what  is  expected  from  the  test,  what  should  be  determined  and  with 

which precision. Power and sample size considerations should also be included; 

•  description  of  test  conditions  (e.g.  size  of  cage,  floor  area,  presence  of  harbourages, 
presence  of  (alternative)  food,  water,  temperature,  photoperiod,  location,  weather 
conditions, season, etc.); 

• 

information  about  acclimatisation  of  the  test  organisms  (occurrence,  conditions,  time 
period); 

•  number of test organisms (sample size); 

•  size of the test population in the field before and after the test; 

•  description  of  the  population  composition  (e.g.  sex,  gravid  or  non-gravid  females, 
nymphs,  larvae,  age  of  the  population  or  generation,  etc.)  if  possible,  in  case  of  field 

 
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trials;  noting  that  the  feeding  behaviour  of  some  insects  (e.g.  cockroaches)  changes 
during their life cycle; 

• 

information  about  starvation  of  the  test  organisms  prior  to  the  test  if  possible  or 
appropriate; 

•  description of the history and origin of the test strain, information whether field strains 

are tested; 

• 

information about all products present in the test, e.g. if, additional to the test product 
an authorised reference product is present; 

•  raw data should be available for each study, rather than just a summary of the results; 

•  show  the  results  of  both  test  (with  biocide)  and  negative  control  (without  biocide) 

treatment, preferably in a table; 

•  description of the monitoring methods used before, during and after the test; 

•  data  analyses  and  statistics  including  a  robust  justification  for  the  choice  of  the  used 

statistical method(s); 

•  any other information that could be considered important affecting the efficacy. 

For  all  products,  efficacy  tests  have  to  be  done  according  to  the  submitted  claim  and  in 
accordance  with  any  applicable  regulations.  In  the  case  of  tests  with  vertebrates,  it  is 
recommended  to  contact  the  prospective  CA  before  conducting  the  test  to  avoid  unnecessary 
animal  testing  due  to  unsuitable  test  designs  or  already  existing  tests.  All  experiments  using 
vertebrate animals shall consider the need to avoid distress and unnecessary pain and suffering 
to experimental animals, in accordance with Directive 2010/63/EU. 

A  clear  claim  should  be  submitted.  The  study  results  should  demonstrate  the  efficacy  of  the 
product according to the label claim and the use conditions, especially the claimed application 
rate.  In  the  case  of  a  roll-on,  lotion,  cream  or  stick  formulation  the  SPC  and  the  label  should 
provide information on the application rate per surface area, e.g. m2 or area of the body. Also, 
for  a  spray  or  a  product  that  is  applied  by  fogging  the  application  rate  and  the  number  of 
sprays or the spray duration per surface area or area of the body should be presented on the 
label and in the SPC. 

For  vapour-based  products,  the  label  should  provide  information  on  the  volume  that  can  be 
treated with the product,  e.g. closet  of x m3, room  of y m3. Vapour-based products may also 
serve as surface repellents, and then shall produce protection of a certain area of surface (m²). 

For  products  applied  as  surface  treatments  a  claim  for  residual  activity  (see  5.6.5.1.4.1.4)  is 
normally valid on the condition that the residues remain undisturbed. This restriction should be 
included  in  the  instructions  for  use  in  the  SPC  and  it  should  be  added  that,  if  applicable  the 
residual  efficacy  can  be  lowered  by  e.g.  cleaning,  degradation  by  light  and  walking.  If  a 
residual  efficacy  after  human  activities  such  as  cleaning  and  walking  is  claimed,  then  these 
procedures must be simulated in the context of efficacy testing. 

Simulated  cleaning  should  follow  the  use  instructions  in  the  SPC  and  may  include  different 
methods such as vacuuming, wet vacuuming, wet or dry cleaning. 

Simulated  walking  is  only  relevant  for  products  to  be  applied  to  floors  in  highly  frequented 
areas. Specific uses stated in the SPC e.g. on couches, mattresses or animal beds have to be 
simulated in the efficacy tests.  

In the case of a change, if the change is in any relevant ingredient influencing the efficacy, e.g. 
adding/removing  preservative  or  change  of  a  food  attractant,  it  should  be  demonstrated  that 
this modification does not influence the product performance. In all other cases, a scientifically 
sound  justification  needs  to  be  provided  that  the  change  does  not  affect  the  efficacy  and  the 
competent authority will then decide if further data is needed. 

5.6.5.1.3.1 Biocidal products containing “known active substances” as co-formulants 

If the product formulation contains as a co-formulant, so-called “known active substance”, e.g. 
certain essential oils, the reasons for the addition of this co-formulant must be explained. The 
applicant should submit a scientifically sound justification, possibly based on scientific literature 
data  on  the  effects  of  the  co-formulant  and  explain  why  it  is  not  an  active  substance  in  the 

 
 
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formulation.  If  the  relevant  information  cannot  be  submitted,  a  study  should  be  submitted 
showing  that  the  efficacy  of  a  formulation  with  the  active  substance  but  without  the  co-
formulant does not differ from the product containing this active substance and co-formulant.35 

5.6.5.1.3.2 Biocidal Product Family (BPF) 

A  BPF  may  contain  several  similar  repellent/attractant  products,  which  could  differ  in  aspects 
such  as  active  substance  content  or  colour.  If  an  application  is  made  for  multiple  products 
based  on  the  same  active  substance  it  may  not  be  required  to  test  all  products  as  long  as 
efficacy  is  demonstrated  for  the  worst-case  product.  For  example,  if  two  products  have 
identical  composition,  but  differ  only  in  the  release  rate  of  the  active  substance,  a  test 
demonstrating  efficacy  of  the  product  with  the  lowest  release  rate  will  also  cover  the  product 
with a higher release rate. If specific/different claims are made for these products with regard 
to the efficacious period, these claims will have to be supported by data.36 

5.6.5.1.3.3 Treated articles 

See section 5.3 for guidance on Treated Articles. 

5.6.5.1.3.4 Test design 

Although internationally or nationally recognised testing methods are  generally preferred, it is 
not  always  possible  to  use  these.  For  some  products  no  standard  methods  are  suitable  or 
available. In that case, a novel test has to be designed. 

Various factors must be considered when designing the tests, for example, the number of test 
individuals  (arthropods,  mammals,  etc.)  needs  to  be  chosen.  The  ultimate  aim  of  relevant 
considerations should be to design experiments that economise on test individuals, but on the 
other hand, generate sufficient power to detect effects of a magnitude considered important to 
demonstrate efficacy. To reduce the overall number of test individuals, replicate tests should be 
conducted.  Another  argument  for  using  replicates  is  to  account  for  the  variation  among  test 
individuals in susceptibility and responses to the biocides. The numbers of test individuals per 
replicate  group  and  dose  level  (treatment  group)  as  well  as  the  number  of  replicates  in  the 
entire study need to be established prior to conducting the tests. As the improvement in power 
reduces substantially as the number of replicates increases beyond five, it is usually sufficient 
to  conduct  five  replicate  tests  at  each  dose  level,  employing  10  (or  20)  test  individuals  each. 
The precise needs will depend on the size of the variances, relative and absolute, between and 
within  the  replicates.  This  can  differ  between  test  organisms  and  test  design.  Sample  size 
should  be  adequate  to  detect  differences  among  groups  (negative  control  vs  treated)  with  a 
statistical power of at least 80%. Some details on these issues are outlined at the end of each 
section.  

Useful information on the principles of test design, analyses end evaluation of efficacy trials can 
be found in the EPPO standards, see Appendix 18. 

5.6.5.1.3.4.1 Test examples 

In  the  respective  sections,  examples  are  given  of  what  kind  of  tests  can  be  expected  for 
efficacy testing. Sometimes these examples are a summary of a standard test, in other cases, 
a company test is described or a general idea of what the test should be like is given. There is 
a great variation in how specific the description is. This is only to give an idea of what kind of 
tests  could  be  provided.  More  detailed  descriptions  of  tests  can  be  found  in  the  standard  test 
methods  (norms)  listed  in  Appendix  18.  This  is  a  list  of  all  available  methods  (according  to 
current  knowledge)  without  distinction  on  suitability,  usefulness,  repeatability,  the  order  of 
acceptability or robustness. Some norms might have a different approach than described in the 
section  for  a  particular  organism.  If  this  approach  is  more  suitable  for  the  product  under 
investigation the norm should be used. 

In  all  cases,  these  tests  are  only  meant  as  examples,  not  obligatory  requirements.  Since 
products  are  so  diverse  in  application  method,  mode  of  action,  etc.  this  guidance  cannot 
possibly  cover  all  testing  possibilities  for  repellents  and  attractants.  Deviations  from  test 

35 CA-Jan18-Doc.4.2_final: Addressing concerns of co-formulants that contribute significantly to a product’s efficacy 

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designs are possible in adoption to the intended uses and must be explained. Applicants should 
design a test protocol and afterwards contact their prospective CA to agree upon it. 

5.6.5.1.3.4.2 Laboratory trials 

Laboratory  studies  are  performed  to  validate  the  efficacy  in  a  laboratory  test  design. 
Laboratory no-choice tests are not required for product authorisation but can be used for active 
substance approval to show an innate level of activity for the active substance and to evaluate 
the  right  dose.  A  simple  choice  test  can  then  be  used  to  see  whether  a  product  is  effective 
under  ideal  conditions.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided. 

For  product  authorisation  purposes,  a  simulated-use  or  field  test  is  normally  required,  see 
section 5.6.5.1.3.4.3). 

5.6.5.1.3.4.3 Simulated-use tests versus field trials 

Simulated-use tests are linked to practical conditions and can, in some cases, be sufficient for 
demonstrating  the  efficacy  and  can  include  factors  like  ageing,  weathering,  UV,  washing,  etc. 
Field  tests  provide  a  good  indication  of  how  the  product  works  in  practice/under  field 
conditions,  to  evaluate  how  the  efficacy  can  be  affected  by  a  variety  of  factors  (the  weather, 
population density, natural fluctuation of the population over time, etc.). 

Simulated-use  tests  or  field  trials  with  the  test  organisms  are  normally  needed  to  assess  the 
efficacy of the product. Field trials are not mandatory in some cases, as outlined in the sections 
on  specific  groups  of  organisms.  In  order  to  eliminate  the  risk  of  disease  transmission  to 
human volunteers in field settings, field trials with repellents against ticks and mosquitoes are 
not  required  for  authorisation  of  products  applied  on  humans  or  clothing.  Pre-existing  studies 
may be submitted as additional information, but field trials are not assessed as key studies for 
these  products.  If  field  trials  are  conducted,  they  have  to  take  place  in  an  area  with  an 
appropriate density of the target organisms and at a time when the relevant target organisms 
are  abundant.  For  testing  on  animals,  field  trials  can  be  accepted.  These  trials  should  be 
conducted on animals that are exposed to the target organisms anyway so that the studies do 
not impose an additional risk of disease transmission. Field tests should preferably take place 
in Europe or other relevant regions according to the claims, e.g. tropical regions. Where testing 
in  Europe  is  not  possible,  the  conditions  and  target  organisms  must  be  confirmed,  and  their 
relevance justified. 

Simulated-use tests, depending on the use of the product, can be conducted in indoor as well 
as in outdoor conditions. In both cases it must be ensured that the tests are conducted under 
controlled  conditions  according  to  the  label  claim  and  the  use  conditions  claimed  in  the  SPC 
(see section 5.6.5.1.4.1.3, further definitions see section 3.1.1 of this guidance). 

In the case  of field trials, a full description  of  any factors that might be expected to influence 
product  performance  including  raw  data  shall  be  given.  These  are  intended  to  provide  the 
authorities with information to assist with the interpretation of the results obtained. These may 
include general levels of sanitation, treatment history, etc. 

5.6.5.1.3.4.4 The importance of negative controls in efficacy studies 

The importance of control experiments for efficacy studies must be stressed with regard to the 
efficacy  evaluation.  Studies  should  be  conducted  alongside  negative  controls  to  provide  a 
reference point for the treatment results. A useful definition of this term is given: “A negative 
control situation may be one in which the experimental design of the study is identical to that 
of  the  biocide  challenge  test  except  that  the  biocidal  product  is  not  applied  or  the  product 
without active substance is applied in the control study.” 

The  negative  control  trial  should  normally  be  of  similar  size,  i.e.  number  of  replicates  as  the 
test itself, to make statistical comparison possible. 

It  is  recognised  that  the  generation  of  such  control  data  can  be  relatively  straightforward  in 
well-defined  test  situations  such  as  laboratory  and  simulated-use  tests.  However,  it  is  also 
recognised that this can present a problem in field situations, where  control  sites may not be 
environmentally equivalent to the treatment site. 

In such instances, there may be an alternative means of generating reference data other than 
collecting  data  from  an  untreated  site.  This  method  may  involve  pre-treatment  monitoring  of 

 
 
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the  site  in  question.  This  monitoring  must  be  quantitative,  e.g.  assessment  of  numbers  of 
trapped insects. In these instances, a “baseline” infestation level would be established through 
such  monitoring  and  then  the  effect  of  treatment  on  this  baseline  can  be  assessed.  Post-
treatment monitoring is required for this method. 

5.6.5.1.3.5 Use conditions for biocidal products applied on clothing or fabrics 

Residual  treatments  may  also  involve  the  claim  “unaffected  by  washing”.  For  the  claim 
“unaffected  by  washing”,  the  label  and  the  SPC  must  indicate  how  often  the  textile  can  be 
washed  without  reducing  the  efficacy  of  the  biocidal  product.  Therefore,  clothes  or  fabrics 
should  be  repeatedly  washed  according  to  the  instructions  for  use.  An  ISO  6330  standard  for 
washing performance assessment should be used where relevant, see Appendix 18. Any claims 
regarding the efficacy after a certain number of washes should be reflected in efficacy testing 
after the corresponding number of standardised washing cycles. 

The efficacy data should be relevant to prove the submitted claim(s). Therefore, the efficacy of 
biocidal products must be proven for the whole test individual that should be protected. If only 
specific  body  areas  intend  to  be  protected,  e.g.  ears,  udders,  etc.  then  the  efficacy  for  the 
claimed area must be proven. 

For  specific  claims,  e.g.  prevention  of  bites  through/prevention  of  bites  next  to  the  treated 
clothes, relevant tests have to be submitted. 

Any  additional  claim  referring  to  wearing  conditions,  such  as  “unaffected  by  UV-light”,  should 
be justified by efficacy data. If the claim includes an area other than the area directly treated 
(“halo effect”), this also needs to be justified by efficacy data. 

5.6.5.1.3.6 Mode of action 

There  are  a  variety  of  modes  of  action  and  possible  effects  on  target  organisms  (arthropods, 
molluscs, leeches, snakes, mammals, birds,  etc.) derived from the proposed use  of a product 
to repel or attract. The available data should give brief details to indicate the route and nature 
of the action, e.g. whether an action is by contact, short or long distance, and the nature of the 
effect,  e.g.  sex-specific  or  unspecific  behaviour,  attraction,  mating  disruption.  If  the  mode  of 
action depends on organisms’ sex or developmental stage the efficacy trials should be designed 
to address the most appropriate sex and/or life cycle stage. 

5.6.5.1.3.7 Attractants in traps 

There are two types of traps which have to be distinguished: traps used to considerably reduce 
or eliminate the population of the pest species, and traps used for monitoring purposes, i.e. to 
detect an infestation. Monitoring traps are not in the scope of this guidance. 

If the product is applied in a trap, the entire product, including the attractant and trap should 
be  tested.  If  authorisation  is  sought  for  different,  but  similar,  kinds  of  traps  one  or  more 
representative traps need to be tested. Variations, e.g. in trap size, shape, colour or material, 
will be allowed without the need of a special approval when a scientific justification is provided 
that  the  change  does  not  impact  efficacy.  The  representative  trap  of  the  final  product  in  the 
application should be as similar as possible to the product to be marketed. 

A trap with an added attractant should catch significantly more individuals than one without the 
attractant. 

5.6.5.1.3.8 Proof of non-insecticidal effect for repellent products 

It has to be proven that a repellent product at the intended uses does not cause any adverse 
killing  effect  to  the  target  organism(s)  and  its  efficacy  comes  from  the  repellent,  not  killing 
effect.  Therefore,  the  mortality  should  be  determined  for  repellent  products  containing  active 
substances approved or under review for both PT18 and 19. For other PT19 active substances, 
that  are  only  approved  or  under  review  for  PT19,  scientific  peer-reviewed  literature  research 
should  be  done  by  the  applicant  in  order  to  verify  if  a  lethal  effect  for  the  applied  target 
organisms  and  product  usages,  i.e.  application  method  and  application  rate  according  to  the 
SPC was observed in studies. The methodology and the outcome of the literature research have 
to be stated in the PAR. If a lethal effect is reported in the literature or no literature research 
has  been  done,  then  tests  should  be  required  in  order  to  demonstrate  that  at  the  claimed 
application rate no lethal effect is observed. 

The requirements for products against invertebrates: 

 
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Mortality  in  the  treatment  group  should  be  similar  to  the  control  group;  if  mortality  in  the 
treatment group will exceed 10%, justification from the applicant is needed. 

An additional monitoring step should be included in a simulated-use test, which is described in 
detail for each target organism in the respective sections. In these  tests, the product must be 
applied according to the instruction of use. Mortality should be compared to a negative control 
group.  Laboratory  tests,  e.g.  filter  papers  in  Petri  dishes,  are  not  sufficient  to  prove  the  non-
insecticidal  effect  of  repellent  products,  because  the  test  conditions  represent  no-choice 
conditions  which  do  not  correspond  to  the  application  of  the  product.  Field  trial  can  only  be 
used  to  prove  the  non-insecticidal  effect  if  the  observed  population  size  can  be  exactly 
determined  before  and  after  the  trial.  However,  any  observed  mortalities  during  field  trials 
should  be  mentioned  in  the  test  summary  if  it  is  possible.  In  addition  to  the  assessment  of 
mortality  in  the  simulated-use  test  or  field  trial  no  further  individual  studies  for  testing  the 
mortality are necessary. 

The study report should contain information on how mortality was evaluated. A clear distinction 
between mortality (criteria for the proof of non-insecticidal effect) and moribund/knock-down is 
necessary for the evaluation. The following definitions can be adapted for the evaluation: 

•  Mortality refers to dead arthropods that do not move, even when poked or probed after 

the specific period of time according to the requested test method. 

•  Moribund  or  knock-down  refers  to  arthropods  that  react  to  stimuli  but  are  unable  to 
move in a coordinated manner, e.g. to upright itself or walk properly. These individuals 
are observed in the following days to verify if they recover or die. 

Products that do not meet these requirements cannot be authorised. 

The requirements for products against vertebrates: 

Mortality due to exposure to the repellent is not acceptable. If some accident occurs during the 
trial and this is justified in the test report it can be acceptable, but mortality in the treatment 
group should be  similar to the  control group and neither group should exceed 10% mortality. 
No additional efficacy studies for testing the mortality are necessary. 

In  any  case,  clinical  monitoring,  e.g.  activity  (grooming,  movement),  weight  gain/loss, 
strength,  ataxia,  posture,  etc.  (for  more  details  see  OECD  guidance  ENV/JM/MONO(2000)7  in 
Appendix  18)  of  the  vertebrate  animals  is  always  required,  at  least  for  laboratory  and 
simulated-use tests, in order to be sure that the product has no adverse side effects. This has 
to be documented in the trial report. 

Products that do not meet these requirements cannot be authorised. 

5.6.5.1.4 Methodology of assessment 

Methods of application 

When  considering  the  overall  evaluation  of  a  proposed  claim  competent  authorities  should 
ensure  that  the  data  presented  are  relevant  not  only  to  biological  challenge  and  treatment 
environment  but  also  that  the  method  of  application  and  application/dose  rate(s)  used  in  the 
test(s) are appropriate to the claims and proposed use of the product. 

The application technique should therefore reflect the claims proposed on the label and in the 
SPC,  whether  it  is  a  topical  application,  application  on  clothing,  general  surface,  crack  and 
crevice or space treatment, spatial repellent, or attractant in traps. 

The  application  method  may  also  describe  a  specific  pattern  of  treatment.  This  is  particularly 
common for spray applications but may also apply to other formulation types.  

General descriptions of some common treatment patterns are given below. 

 
 
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Surface treatments 

These  are  treatments  where  the  product  is  applied  on  surfaces  such  as  walls,  floors  and 
ceilings,  or  used  as  a  treatment  on  outdoor  surfaces.  These  treatments  may  involve  the 
treatment of a large area of surface or may only involve an application to a narrow band. 

Surface treatments can also include application to temporary or permanent bodies of water, 
e.g. in mosquito control or to solid and semi-solid manure. 

Crack and crevice treatments 

These  are  treatments  where  products  are  applied  into  cracks  and  crevices  where  insects 
hide  and  harbourage,  or  through  which  they  may  enter  the  building.  Such  openings 
commonly  occur  at  expansion  joints,  between  different  elements  of  construction  and 
between  equipment  and  floors.  These  openings  may  lead  to  voids  such  as  hollow  walls, 
equipment legs and bases, conduits and junction or switch boxes. 

Space treatments/spatial repellents 

These  are  treatments  where  the  product  is  applied  to  the  air  rather  than  onto  a  surface. 
They are intended to disperse small droplets  or particles into the  atmosphere of a room or 
other  open  space,  where  they  will  normally  stay  for  a  period  of  time  (very  small  particles 
may stay in the air for several hours under still conditions). 

Spot treatments 

These are treatments where products are applied to limited areas on which pests are likely 
to occur. These areas may occur on floors, walls and bases or undersides of equipment. 

General considerations 

The  efficacy  data  submitted  should  demonstrate  that  the  biocidal  product,  when  used  as 
directed by the product label and the SPC, will result in a measurable beneficial effect. The data 
supplied should demonstrate that an acceptable, consistent level and duration of the intended 
effect will result from the use of the product at the recommended dose rate. 

This  may,  depending  on  the  individual  product,  be  measured  as  a  reduction,  repellency  or 
attraction  of  the  pest  population  to  an  acceptable  level.  The  acceptable  level  may  vary 
depending on the purpose of the proposed use. 

Competent authorities should evaluate available data to determine whether they are sufficient 
to support the proposed claim. 

The  competent  authority  will  examine  the  submitted  data  package  and  a  judgement  will  be 
made  as  to  whether  any  data  omissions  are  considered  significant  enough  to  prevent  the 
conclusion that efficacy is proven. Identified data omissions will be communicated back to the 
applicant. The applicant can then supply additional data or modify their proposed claims in line 
with whatever has been sufficiently supported. 

Any  known  limitations  on  efficacy  should  be  considered  during  the  assessment  and  the 
following actions taken as necessary: 

•  State  possible  restrictions  or  recommendations  concerning  the  use  of  the  product  in 

specific environmental or other conditions; 

•  State  possible  factors  that  can  reduce  the  efficacy,  for  instance  hot,  cold  or  humid 
environments or the presence of other substances, in addition to the grounds for these; 

•  Information  on  resistance,  the  likelihood  of  its  development,  e.g.  scientific  literature 
research,  and  if  needed  appropriate  resistance  management  strategies  should  be 
provided.  Resistance  management  should  be  addressed  if  appropriate  guidelines  are 
available,  even  if  it  is  not  to  be  expected  that  resistance  will  build  up  for  repellents 
and/or attractants; 

•  State if the product cannot be mixed with, for example, other biocidal products; 

•  State if the use of the product with other biocidal products is recommended. 

5.6.5.1.4.1 Assessment of specific claims 

Sometimes a claim will include specific properties of the product, e.g.: 

 
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•  repels ticks for 5 hours; 

•  residual effect up to 3 months; 

•  storage period up to 5 years; 

•  repels mosquitoes in tropical regions. 

Where a particular property is claimed the data submitted to support the product should show 
that  the  product  actually  has  these  properties.  If  data  do  not  support  this  claim,  the  product 
may  still  gain  authorisation  with  amended  claims,  provided  that  the  product  still  shows 
acceptable efficacy. 

For example: If a product claims a protection time of 5 hours against ticks, the data submitted 
must show a repellency for 5 hours after application in order for these claims to be acceptable. 
However, if the submitted data showed repellency only for 4 hours, the product may still gain 
authorisation provided that the product claims were amended to “protection time against ticks 
for 4 hours after application”. 

Situations  such  as  the  example  above  will  require  each  study  to  be  evaluated  on  its  own 
merits, taking into account what the data are actually showing. Evaluators must use scientific 
judgement to determine when authorisation would not be acceptable. 

5.6.5.1.4.1.1 Claims with respect to target organisms 

Broad claims, such as "crawling insects" or "flying insects", should normally not be supported, 
as  such  claims  would  cover  a  diverse  range  of  organisms’  habitats,  morphology,  biology  and 
behaviour.  Specific  information  on  the  test  species  for  specific  organisms  are  given  in  the 
following sections.  

In  the  European  tropical  overseas  regions,  the  most  common  genus  encountered  could  be 
different.  A  specific  claim  should  therefore  be  proposed,  with  referred  target  organisms.  This 
special request could concern for example special mosquito or cockroach species. 

For  claims  with  different  target  organisms,  e.g.  cockroaches  and  ants,  efficacy  data  for  each 
target organism in line with the required species in the specific sections must be provided. 

5.6.5.1.4.1.2 Claims relating to storage of a product 

For a stated storage period claimed up to two years no tests with aged products are necessary 
if  the  physical/chemical  analysis  ensures  that  the  product  composition  is  still  stable  after 
storage and that the active substance content has not decreased more than 10%. 

When a product is claimed to be effective after storage of more than two years, it is necessary 
to  demonstrate  by  analytical  studies  that  the  product  composition  is  still  stable  and  that  the 
active  substance  content  has  not  decreased  more  than  10%.  If  the  degradation  of  the  active 
content  is  >  10%  the  applicant  needs  to  submit  data  for  effectiveness  against  all  target 
organisms in a simulated-use test with the product stored at ambient temperature at the end 
of  the  maximum  storage  period.  Accelerated  ageing  studies,  in  which  the  product  tested  is 
stored  under  challenging  conditions,  are  not  acceptable  as  these  cannot  simulate  longer 
storage periods.  

For  products  intended  for  use  as  attractant  in  combination  with  PT18  (bait  products),  the 
efficacy evaluation should be done on the basis of the requirements for bait products37.  

In applications following the simplified authorisation procedure storage stability must either be 
proven in a storage stability test or in an efficacy test at the end of the stated storage period. 

5.6.5.1.4.1.3 Claims relating to outdoor use 

When products are intended for outdoor use, e.g. for surface or space treatment or attractants 
in  traps,  tests  should  normally  demonstrate  efficacy  under  outdoor  conditions.  Changes  in 
temperature  and  rainfall  can  have  an  effect  on  the  efficacy  of  the  products.  In  general,  field 
trials cover this outdoor use. Treated surfaces or products and their negative controls have to 
be kept outside to ensure adequate weathering and the outdoor conditions must be specified, 
i.e. temperature, humidity, photoperiod. 

37 See Technical Agreements for Biocides - Shelf life of PT18 bait products 

 
 
 
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For  specific  uses  like  “in  porches”  the  conditions  in  the  efficacy  test  should  match  with  the 
intended use of the product. 

If  both  “indoor”  and  “outdoor”  uses  are  claimed  for  a  product,  then  efficacy  studies  under 
outdoor  conditions  can  be  used  for  authorisation  of  the  claim  “indoor  use”,  when  use 
instructions  (like  dose,  application  rates,  frequency)  are  identical.  However,  efficacy  studies 
under indoor conditions cannot be used for authorisation of the claim "outdoor use" (exception 
for mosquitoes and ticks, for details see 5.6.5.1.3.4.3). 

Weathering conditions for surface treatment with outdoor usage 

Residual efficacy for products with an intended outdoor use should be tested in simulated-use 
tests or field tests.  

When  usage  in  tropical  regions  is  claimed  in  simulated-use  tests  then  environmental 
parameters (temperature, humidity, rainfall, solar radiation, etc.) should be adapted to tropical 
regions. Field tests should preferably take place in tropical regions. 

Different  treated  surfaces  (according  to  the  claim;  e.g.  ceramic  tile,  plywood,  stainless  steel, 
concrete) should be exposed to (unless stated otherwise on the label) the following conditions: 

•  air temperature during storage period must be in the range of 19-29°C; 

• 

rainfall,  if  necessary  this  could  be  mimicked  by  artificial  watering  (examples  for  a 
harmonized  protocol  e.g.  from  material  testing  for  clothing  (EN14360)  or  wood 
preservatives  (Annex  F  of  EN152),  see  Appendix  18)  for  at  least  20%  of  the  residual 
efficacy period (for specific claims e.g. ‘rainfast 1hr after application’ or ‘water resistant’ 
additional testing is considered); 

•  direct  sunlight  for  at  least  30%  of  the  residual  efficacy  period  as  most  products  with 
outdoor  use  will  typically  be  applied  during  the  summer  months  when  sunlight  is 
considerable  for  the  majority  of  the  day  (6  to  12  hours  per  day;  may  depend  on  the 
claimed region to be used). 

Deviation  from  these  norms  is  possible  but  should  be  justified  in  the  application,  e.g.  usage 
under a roof. 

5.6.5.1.4.1.4 Claims for residual efficacy 

Some  pest  management  programmes  involve  the  use  of  relatively  stable  active  substances 
applied  to  buildings,  surfaces  or  traps  to  leave  residual  deposits.  These  compounds  are 
intended  to  remain  chemically  active  and  therefore  effective  for  periods  of  weeks  and  up  to 
several months following treatment, i.e. they have a high residuality. Residual life is a term to 
describe  the  period  during  which  the  biocide  will  be  present  in  sufficient  quantity  to  affect  a 
target organism. 

Residual efficacy must be proven in tests. Usually, testing is performed to establish the efficacy 
directly after application and at the end of the residual life of the product. 

The types of surfaces to which residual products are applied must be reported since the surface 
type has a pronounced effect on the amount of active residue available to the target organism. 
In  general,  two  porous,  e.g.  unpainted  wood,  carpet,  concrete,  linoleum  and  one  non-porous 
surface,  e.g.  stainless  steel,  ceramic  tile,  vinyl  tile,  varnished  wood  should  be  tested.  The 
efficacy of each surface should be proven in a separate test, i.e. three tests for three different 
surfaces. 

For  outdoor  use  for  surface  applications,  information  about  exposure  time  of  the  treated 
surface to sunlight has to be provided. 

Efficacy  data  submitted  to  the  competent  authority  in  support  for  residual  treatments  should 
indicate the appropriate dosage and the utility of the formulation when used as directed. 

The  duration  of  efficacy,  e.g.  complete  protection  time  of  repellent  applied  on  skin/clothes, 
residual  efficacy  for  surface  treatment  demonstrated  in  efficacy  tests  should  be  stated  in  the 
SPC and on the label. 

5.6.5.1.5 Definitions to determine Complete Protection Time (CPT) 

For  claiming  a  CPT,  the  following  definitions  are  of  special  importance  in  understanding  this 
guidance.  They  apply  only  in  the  context  of  this  guidance  for  products  for  which  a  CPT  is 

 
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required (see e.g. 5.6.5.3 Bed bugs, 5.6.5.6 Fleas, 5.6.5.9 Mosquitoes, 5.6.5.13 Ticks) and are 
not intended to be more generally applicable. 

To determine the  CPT the target  organisms  should be exposed to the product  every 30  or 60 
minutes with the first exposure being 30 minutes after the product application. 

Complete Protection Time is the time from the application of a repellent until the last effective 
observation,  before  the  efficacy  failure  by  a  confirmed  event.  The  CPT  to  be  specified 
corresponds to the time interval before the confirmed event, i.e. the time period in which the 
product achieves complete repellency against the target organism. For example, at 30 minutes 
observation  intervals,  the  first  confirmed  event  occurs  4  hours  after  product  application, 
confirmed  by  the  second  event  after  4.5  hours  after  product  application.  Then  the  CPT  of  the 
product can be claimed for 3.5 hours. At 60 minutes observation intervals, the first confirmed 
event  occurs  4  hours  after  product  application,  confirmed  by  the  second  event  after  5  hours 
after product application. Then the CPT of the product can be claimed for 3 hours. 

A confirmed event is one target organism that is not repelled followed by another similar event 
within the same or the next exposure period, i.e. 30 or 60 minutes. The first event is confirmed 
by the second; the second event is the confirming event. In  the case where one non-repelled 
target  organism  is  not  followed  by  another  one  in  the  same  or  the  consecutive  test  interval, 
then efficacy testing will continue. 

An unconfirmed event is one target organism that is not repelled and not followed by another 
similar event within the same or the next exposure period, i.e. 30 or 60 minutes. 

For  mosquitoes,  a  landing  is  the  act  of  a  flying  mosquito  alighting  on  human  skin  without 
probing or biting. Landing is not always associated with probing. Since a repellent may provide 
efficacy by a reduction in biting activity but not in landing, this event does not indicate a failure 
of  repellent  efficacy.  Furthermore,  some  products  do  not  prevent  landing,  because  the  active 
substances are only effective by direct contact. 

The following events indicate a failure of repellent efficacy by testing with mosquitoes: 

Probing is the act of penetrating human skin by the mouthparts of a mosquito without ingestion 
of blood. 

Biting is the act of penetrating human skin by the mouthparts of a mosquito with ingestion of 
blood, typically associated with abdominal swelling and colour change. 

CPT calculation 

The dataset with CPT for each volunteer should be tested for normal distribution. When results 
are  normally  distributed,  it  may  be  appropriate  to  report  the  mean  CPT  across  all  treated 
subjects with its standard error. Any right-censorship of repellency data, e.g. for one volunteer, 
the product is  effective through the entire  observation time, will lead to skewed estimation of 
both the mean and the variance around it. The use of the median CPT with its 95% confidence 
limits  as  the  summary  measure  of  CPT  is  recommended,  if  right-censorship  occurs,  see 
Appendix 18: OPPTS 810.3700. 

When the data do not fit a normal distribution—more typical of repellency datasets—it may be 
possible to transform them to fit a distribution for which a parametric method  of analysis can 
be  employed.  When  the  data  do  not  fit  and  cannot  be  transformed  to  fit  an  underlying 
distribution, non-parametric analyses, such as Kaplan-Meier survival analysis, are suggested. 

5.6.5.1.6 Assessment of authorisation 

When  considering  the  overall  evaluation  of  proposed  claims,  competent  authorities  should 
ensure that the data and the method of application and application/dose rates used in the tests 
are appropriate to the claims and proposed use of the product. 

5.6.5.1.6.1 Norms and criteria 

The  test  results  are  compared  directly  with  the  norms  and  criteria  for  efficacy  described  per 
organism in the respective sections. The performance criteria set in this guidance ask for high 
levels of efficacy. However, products that do not fully meet the criteria can still be valuable in 
some cases. 

When a product does not perform to the criteria it should be justified in the application why this 
product  is  still  recommended  for  authorisation.  In  a  field  trial  the  criteria  may  not  be  met 

 
 
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because  of  immigration  of  insects  from  untreated  areas,  e.g.  flies,  mosquitoes.  When  this  is 
explained well in a justification the product might still be accepted for authorisation, depending 
on the results of other field and simulated-use trials. 

It  should  be  taken  care  of  that  no  placebos  or  misleading  products  are  authorised.  If  the 
efficacy  level  is  significantly  lower  than  the  criteria  stated  it  should  be  mentioned  in  the  SPC 
and  on  the  label  e.g.  use  as  Integrated  Pest  Management,  other  biocidal  products  used  in 
combination.  The  justification  will  be  evaluated  case  by  case.  The  product  should  not  be 
authorised unless there is a good reason for having a product of lower effectiveness. 

5.6.5.1.6.2 Assessment 

The  assessor/expert  assesses  on  the  basis  of  the  claim  and  the  above  criteria.  If  the  product 
was assessed to be sufficiently effective in simulated-use and/or field tests, it will be authorised 
as far as efficacy is concerned. 

5.6.5.2 Ants  

5.6.5.2.1 Introduction  

Ants may cause inconvenience both indoors and outdoors.  

In Europe the following ant species are common:  

Black garden ant 

Lasius niger or other Lasius species 

Pavement ant  

Tetramorium caespitum  

Red ant  

Myrmica rubra  

Erratic ant  

Tapinoma erraticum  

Carpenter ants  

Camponotus spp. 

Next to these native ant species, introduced tropical ants can cause problems, mainly indoors.  

Of the tropical and invasive ant species, there are four species that are most commonly found 
causing inconvenience in buildings in Europe:  

Pharaoh ant  

Monomorium pharaonis  

Argentine ant  

Linepithema humile 

Ghost ant  

Tapinoma melanocephalum 

Invasive garden ant  Lasius neglectus 

This is a non-exhaustive list; other species may be tested if the applicant wishes to claim for a 
specific target organism. 

5.6.5.2.1.1 Biology  

Ant  development  involves  a  complete  metamorphosis  that  includes  distinct  egg,  larval,  pupal 
and  adult  stages.  Most  ant  species  form  colonies  comprised  of  complicated  social  structures 
that  include  infertile  female  workers,  one  or  more  specialised  fertile  winged  queen(s)  and  (at 
certain  stages  in  nest  development)  sexually  mature  winged  males.  Some  species  have 
developed  additional  specialised  workers  that  are  responsible  for  guarding  the  nest  and 
attacking  intruders,  whilst  others  perform  domestic  and  foraging  duties.  These  workers  will 
actively  forage  on  a  wide  range  of  foods  including  sweet  substances,  seeds,  insects,  aphid 
secretions, meat, blood and other protein sources. A successful foraging ant also has the ability 
to  communicate  where  to  find  food  to  its  co-workers,  using  chemical  signals  (trail 
pheromones).  The  control  of  tropical  and  invasive  ants  can  be  complicated  by  their  peculiar 
colony  system:  These  species  are  polygynous,  meaning  each  colony  contains  many  queens. 
Hence,  a  colony  can  quickly  fragment  into  several  colonies,  and  colonies  do  not  show 
aggressive behaviour against each other (unicoloniality) due to a lacking nestmate recognition.  

Many native ant species use sand or soil to build their nests, but some also use wood, decaying 
wood or insulation materials. Some carry water into the nest to keep the  right humidity. Such 
nest-building habits may occasionally cause harm to wooden constructions or insulation. 

5.6.5.2.2 Dossier requirements  

Dossier requirements are stated in the Introduction, see section 5.6.5.1.3. 

 
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5.6.5.2.2.1 Test species  

For  a  general  claim  “against  ants”,  at  least  two  ant  species  each  belonging  to  different  ant 
subfamilies (Formicinae, Myrmecinae or Dolichoderinae) have to be tested. For a general claim 
“against tropical ants”, at least two tropical ant species of at least two different ant subfamilies 
(Formicinae, Myrmecinae or Dolichoderinae) have to be tested. For a species- or genus-specific 
claim, testing against the claimed species or genus is required. 

Due to the specificity of certain active substances, e.g. pheromones, for products based on an 
active  substance  with  a  species-specific  mode  of  action,  only  effects  against  ant  species  that 
have been tested under simulated-use and/or field conditions, depending on the type of claim, 
should be claimed on the product label and in the SPC. 

National  and  communal  laws  might  have  to  be  regarded  during  the  evaluation  for  the  use  of 
products against some species such as for example pharaoh ants (M. pharaonis). 

5.6.5.2.2.2 Requirements per type of claim and test methods 

A  laboratory  test  is  not  required  for  product  authorisation  (exception  see  5.6.5.2.2.2.1 
Repellent products intended for use as surface treatment) but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided. 

5.6.5.2.2.2.1 Repellent products intended for use as surface treatment 

For product authorisation purposes the efficacy of repellent products should be proven in: 

•  a  simulated-use  test  according  to  the  instruction  for  use  (test  design  example  see 

5.6.5.2.2.2.1.2), or 

•  a  field  trial  according  to  the  instruction  for  use  (test  design  example  see 
5.6.5.2.2.2.1.3)  and  additionally  a  laboratory  trial  testing  the  required  different 
surfaces. 

Products applied onto surfaces may act either by evaporation or on the surface itself. 

For  products  applied  on  surfaces,  two  porous  and  one  non-porous  surface  should  be  tested, 
e.g. ceramic tile, plywood, painted plywood, stainless  steel,  unpainted wood, carpet,  concrete 
for a general claim as “surface treatment”.  The efficacy of each surface should be proven in a 
separate test, i.e. three tests for three different surfaces. For authorisation of a product to be 
used on a specific type of surface the efficacy for only this specific surface should be tested. 

For residual efficacy, ants are exposed to the product at several time intervals after application 
(including the end of the claimed period). 

If  outdoor  use  is  claimed,  the  treated  surface/substrate  or  repellent  product  and  control 
surfaces/substrates  have  to  be  kept  outside  to  ensure  adequate  weathering  and  the  outdoor 
conditions  must  be  specified  (for  detailed  information  regarding  the  requirements  see 
Introduction, section 5.6.5.1.4.1.3). 

Proposed  claims  regarding  the  performance  of  the  product  should  be  simulated  in  the  study. 
For  example,  for  the  claim  “unaffected  by  cleaning/vacuuming”,  the  surface  should  be 
repeatedly cleaned during the trial (for details see Introduction, section 5.6.5.1.3). 

The repellents used for the test should be identical to the product to be marketed. 

5.6.5.2.2.2.1.1 Laboratory test  

A  choice  or  a  no-choice  test  can  be  used,  see  Appendix  18:  Krüger  A.,  Knobelspieß  S.,  and 
Schmolz E.. 

General set-up: At least 50 worker ants are placed within a Petri dish, which is standing on a 
beaker  surrounded  by  water,  building  an  artificial  “island”.  Ants  can  leave  the  island  via 
one/two bridge(s) ending in two separate beakers, see Figure 12. Other possibilities to escape 
should be blocked by a slick insect barrier, e.g. fluon. 

Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative  control) 
should be performed. 

 
 
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Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  surfaces/substrates  or  repellent  product  (temperature,  humidity,  ventilation,  and 
photoperiod). 

Choice test 

Ants can leave the island via two bridges of the same material. The formulated product (spray, 
liquid, gel, powder, dust, etc.) is applied in the middle of one bridge, over the entire width as a 
stripe;  the  second  bridge  remains  untreated.  Typically,  the  stripe  should  be  1  cm  wide  or 
according to the claim. 

Figure 12: Example for the experimental set-up of the laboratory choice test for repellents against ants. 

Within the investigation period, e.g. 30 min., the number of ants crossing more than half of the 
untreated  or  the  treated  bridge,  by  crossing  the  applied  product,  is  recorded.  Subsequently, 
ants  that  crossed  the  middle  of  the  bridge  are  transferred  softly  with  a  paintbrush  into  the 
associated beaker at the end of the bridge. To exclude bias due to pheromone trails in the Petri 
dish  the  bridges  should  be  swapped  among  each  other  after  half  of  the  ants  crossing  the 
bridges. 

To exclude side preference effects, a control treatment without active substance on any bridge 
should be conducted with insects from the same insect population.  

No-choice test 

Ants  can  leave  the  island  only  via  one  bridge.  The  formulated  product  (spray,  liquid,  gel, 
powder,  dust,  etc.)  is  applied  in  the  middle  of  the  bridge,  over  the  entire  width  as  a  stripe. 
Typically, the stripe should be 1 cm wide or according to the claim. 

Within the investigation period, e.g. 30 min., the number of ants crossing more than half of the 
treated  bridge,  by  crossing  the  applied  product,  is  recorded.  In  this  test  set-up  some  ant 
species will have to be encouraged to move away from the Petri dish and  onto the bridge (by 
gentle prodding with a soft brush). Repelled ants must be removed from the test system to be 
able to distinguish clearly the repelled ants from non-running ants. 

5.6.5.2.2.2.1.2 Simulated-use test 

Mandatory requirements: 

•  A choice test with complete nests should be performed in a test arena of at least 600 cm2 
total  volume  (use  of  two  small  arenas  is  possible),  see  Appendix  18:  Krüger  A., 
Knobelspieß S. and Schmolz E.. 

•  Replicates:  A  minimum  of  3  independent  replicates  (each  treatment  and  negative 

control) should be performed.  

•  Each replicate consists of an ant nest, containing workers, brood and queen(s). In case 

of tropical ants at least 2 queens and 200 workers per colony should be used. 

 
 
 
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•  Environmental conditions must be specified for the test itself and during the storage of 
repellent  product  (temperature,  humidity, 

treated  substrates/surfaces  or 

the 
photoperiod, ventilation).  

•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8). 

Test design: The simulated-use test is designed to mimic the practical use situation. Other test 
designs than the following example can be accepted if the protocol is scientifically valid. 

Ant  nests  should  be  introduced  into  arenas.  Arenas  should  be  divided  into  two  similar 
compartments  (Figure  13)  by  at  least  a  1  cm  wide  line  of  slick  insect  barrier  (e.g.  fluon). 
Alternatively, the ant nest is connected via a bridge to  another compartment. Food and water 
are  always  placed  in  the  compartments  without  the  nest.  During  acclimatisation  (at  least  7 
days)  ants  can  reach  the  food  via  one  bridge  and  build  a  pheromone  trail.  According  to  the 
claim the bridges should be of porous and/or non-porous substrate, e.g. ceramic tile, plywood, 
painted plywood, stainless steel, concrete. 

After acclimatisation, the product (spray, liquid, gel, powder, dust, etc.) is applied in the middle 
of the bridge at the recommended label rate(s) following the product use instructions, covering 
the entire width in a stripe. Typically, the stripe should be 1 cm wide or according to the claim. 
Additionally, a  second  bridge of the same material is provided as negative  control. Therefore, 
ants  have  a  choice  of  two  paths  from  the  nest  to  food  and  water,  one  which  they  are 
accustomed to that is now treated, or a new path (Figure 13B). For residual efficacy, ants are 
exposed to the product at several time intervals after application on the bridges (including the 
end of the claimed period). 

Depending on the size of the ant nest and the ant activity for 1 - 5 minutes (longer observation 
periods  are  possible  if  ant  activity  is  low),  the  number  of  ants  traveling  more  than  halfway 
across  the  treated  bridge  (over  the  treated  surface)  and  the  control  bridge  are  recorded 
separately. 

Figure 13: Example for the experimental set-up (A: before and B: after product application) of the simulated-use test 
for repellents against ants. 

Proof  of  non-insecticidal  efficacy:  After  each  test  run  a  defined  number  of  ants  (approximate 
100  workers)  should  be  transferred  into  a  separate  arena  to  monitor  the  mortality  of  the 
workers. 

 
 
 
 
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Efficacy assessment: The potential repellent effect of the product is determined by  comparing 
the number of ants crossing the treated bridge vs. the control bridge.  

5.6.5.2.2.2.1.3 Field trials 

In the  field trials, the product is tested in the  actual  use  situation, for instance in an infested 
home or garden and applied according to the direction  for use in the  SPC.  Other test designs 
than the following example can be accepted if the protocol is scientifically valid. 

Test  design:  The  efficacy  tests  against  ants  should  normally  be  performed  on  a  minimum  of 
three objects with sufficient ant activity (Lasius niger 50 ants within 2 min) in each object. The 
distance between the objects must be large enough to be sure that three different nests will be 
observed. For the most ant species three different infested homes or gardens can be used. For 
field  trials  with  unicolonial  ant  species  (see  5.6.5.2.1)  no  directly  neighboured  objects  should 
be  used  to  avoid  the  testing  of  only  one  supercolony.  An  object  can  be  a  place  in  or  near  a 
building,  where  ants  cause  inconvenience  for  the  inhabitants.  This  may  be  in  a  house,  on  a 
balcony, or on a terrace, depending on the field of use of the product. If the test is performed 
outdoors,  or  ants  are  expected  to  enter  the  treated  space  from  outdoors,  temperature  and 
rainfall  shall  be  recorded.  The  product  should  be  applied  at  the  recommended  application 
rate(s) following the product use instructions. 

A food source can be introduced near the  nest, e.g. 1 meter, for a few days to create an ant 
trail. To ensure consistent attractiveness, the quality and quantity of the food source must be 
maintained throughout the trial. A similar neighbour nest must be tested with a food source as 
a negative control as well. Often ants decide for longer or shorter time periods to neglect food 
sources,  e.g.  if  aphids  occur  or  the  ants  focus  on  protein  collection.  Before  application  of  the 
product  the  time  to  observe  100  ants  must  be  recorded.  This  observation  period  is  used  to 
monitor the ant activity after the product application at three different time points. 

Monitoring  should  be  conducted  at  the  same  locations  (as  the  pre-treatment)  and  at  similar 
times during the entire trial, e.g. at 12.30, 13.00, etc. Monitoring should continue, e.g. 1 day 
after  treatment,  1  week  after  treatment,  etc.  at  least  once  weekly  according  to  the  period 
claimed  on  the  label  and  in  the  SPC.  A  neighbouring  nest  (similar  in  size  and  ant  activity)  as 
negative control must be monitored at the same time to document sufficient ant activity.  

Replicates: A minimum of 3 independent objects (each treatment and negative control) should 
be performed.  

Environmental  conditions  must  be  specified  for  the  test  itself,  and  during  the  storage  of  the 
treated  surface/substrate  or 
repellent  product  (temperature,  humidity,  photoperiod, 
ventilation). Timing of trials should be done according to the ant species biology and life cycle.  
All objects (treatment and control) should be tested during the same period of time and region 
with  a  similar  ant  activity  to  ensure  that  tests  have  not  been  conducted  during  the  natural 
decline in ant activity. 

Efficacy assessment:  The potential repellent effect of the product is determined by  comparing 
the  number  of  ants  in  the  treatment  group  and  the  negative  control  group  monitored  during 
the  observation  period  or  by  comparing  the  number  of  ants  in  the  treatment  group  and  the 
number of ants before the treatment. 

5.6.5.2.2.2.2 Attractants without PT18 active substances  

For  product  authorisation  purposes  the  efficacy  of  attractants  in  traps  without  PT18  active 
substance should be proven in: 

•  a  simulated-use  test  according  to  the  instruction  for  use  (test  design  example  see 

5.6.5.2.2.2.2.1), or 

•  a  field  trial  according  to  the  instruction  for  use  (test  design  example  see 

5.6.5.2.2.2.2.2). 

Traps  should  be  tested  on  their  own,  with  a  negative  control  tested  separately  in  an  identical 
test setting. 

The attractants, e.g. the trap, used for the test should be as similar as possible to the product 
to be marketed (for details see General Introduction, chapter 5.6.5.1.3.7).  

 
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If  outdoor  use  is  claimed,  the  test  should  be  performed  outdoors  and  the  attractant  product 
and control have to be kept outside to ensure adequate weathering and the outdoor conditions 
must be specified (for details see Introduction, section 5.6.5.1.4.1.3). 

5.6.5.2.2.2.2.1 Simulated-use test 

Mandatory requirements: 

•  A choice test with complete nests should be performed in a test arena of at least 600 cm2 

total volume (use of two small arenas is possible). 

•  Replicates:  A  minimum  of  3  independent  replicates  (each  treatment  and  negative 

control) should be performed. 

•  Each replicate consists of an ant nest, containing workers, brood and queen(s). In case 

of tropical ants at least 2 queens and 200 workers per colony should be used. 

•  Environmental conditions must be specified for the test itself and during storage of the 

attractant product (temperature, humidity, photoperiod, ventilation). 

Test design: The simulated-use test is designed to mimic the practical use situation. Other test 
designs than the following example can be accepted if the protocol is scientifically valid. 

Ant nests should be introduced into arenas. The nest is placed in one half of the arena and food 
and  water  are  always  placed  in  the  other  half  of  the  arena.  After  acclimatisation  (at  least  7 
days)  the  attractant  product/trap  is  applied  according  to  the  claim  in  the  half  of  the  arena 
containing food and water. 

The number of trapped ants is monitored at defined time intervals until the end of the claimed 
efficacy  period,  e.g.  24  hours  after  introduction  of  the  product.  For  residual  efficacy,  ants  are 
exposed  to  the  product  at  several  time  intervals  after  application  (including  the  end  of  the 
claimed period). 

Efficacy  assessment:  The  attraction  of  a  product  is  determined  by  comparing  the  number  of 
ants  trapped  with  the  product  vs.  the  control  trap  to  calculate  a  ratio  (product:  negative 
control) of trapped ants. 

5.6.5.2.2.2.2.2 Field trial 

In the  field trials, the product is tested in the  actual use  situation,  for instance in an infested 
home or garden, and applied according to the instruction for use in the SPC. Other test designs 
than the following example can be accepted if the protocol is scientifically valid. 

Test  design:  The  efficacy  tests  against  ants  should  normally  be  performed  on  a  minimum  of 
three objects with sufficient ant activity (L. niger 50 ants within 2 min) in each object. For the 
most  ant  species  three  different  infested  homes  or  gardens  can  be  used.  For  field  trials  with 
unicolonial ant species (see 5.6.5.2.1) no directly neighboured objects should be used to avoid 
the testing of only one supercolony. An object can be a place in or near a building, where ants 
cause inconvenience for the inhabitants. This may be in a house, on a balcony, or on a terrace, 
depending  on  the  field  of  use  of  the  product.  If  the  test  is  performed  outdoors,  or  ants  are 
expected to enter the treated space from outdoors, temperature and rainfall shall be recorded. 
The  product  should  be  applied  at  the  recommended  label  rate(s)  following  the  product  use 
instructions.  

In the field trial, only those animals that contribute to the infestation in the object are included 
in  the  evaluation  of  effectiveness.  Only  for  a  claim  "effective  against  ant  nests"  the  efficacy 
against the whole ant nest must be proven. 

Any bycatch of non-target insects has to be recorded and identified at least to order, preferably 
to the family level. 

Replicates: A minimum of 3 independent objects (each treatment and negative control) should 
be performed 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  storage  of  the 
attractant product (temperature, humidity, photoperiod, ventilation). Timing of trials should be 
done  according  to  the  ant  species  biology  and  life  cycle.  All  objects  (treatment  and  control) 
should be tested during the same period of time and region to ensure that tests have not been 
conducted during the natural decline in ant activity. 

 
 
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Efficacy  assessment:  The  attraction  of  a  product  is  determined  by  comparing  the  number  of 
ants  trapped  with  the  product  vs.  the  control  trap  to  calculate  a  ratio  (product:  negative 
control) of trapped ants. 

5.6.5.2.2.2.3 Attractants in PT18 bait products 

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for  bait 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods). 

5.6.5.2.3 Assessment of authorisation  

5.6.5.2.3.1 Norms and criteria  

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for ants in the following way: 

Products intended for use as repellent: 

Non-insecticidal  efficacy  has  to  be  proven  in  a  simulated-use  test  if  it  cannot  be  waived  (for 
details see Introduction, section 5.6.5.1.3.8).  

The  required  results  for  the  different  tests,  i.e.  laboratory  tests  (not  required  for  product 
authorisation), simulated-use tests, field trials are: 

•  ≥ 90% repellency within the test period (or according to the claim), from the beginning 

and until the end of the claimed efficacy period. 

Attractants without PT18 active substances: 

The  required  results  for  the  different  tests,  i.e.  laboratory  tests  (not  required  for  product 
authorisation), simulated-use tests, field trials are: 

•  at least a ratio of 4:1 of ants trapped in the trap with attractant compared to the control 

trap within the test period (or according to the claim), from the beginning and until the 
end of the claimed efficacy period;  

•  ≥ 80% of the estimated population size trapped within the test period (or according to 

the claim) compared to the negative control), from the beginning and until the end of 
the claimed efficacy period. 

In general  for all PT19  products  claiming  "effective against ant nests" the efficacy against the 
whole ant nest must be proven. 

5.6.5.3 Bed bugs 

5.6.5.3.1 Introduction  

Bed  bugs  are  small,  wingless  blood-feeding  insects.  In  temperate  climate  regions  of  the  EU, 
Cimex  lectularius  is  the  dominant  species.  Cimex  lectularius  and  Cimex  hemipterus  are  the 
most  relevant  species  that  feed  on  humans.  Bed  bugs  are  not  known  to  transmit  diseases  in 
Europe,  but  even  when  they  are  no  pathogen  vectors,  bed  bugs  can  have  significant 
consequences  on  the  health  and  the  quality  of  people’s  lives.  Bites  can  cause  skin  irritation, 
itching  and  skin  lesions.  When  the  presence  of  bed  bugs  is  reported,  it  is  necessary  to  act 
quickly  by  calling  upon  an  exterminator  to  destroy/kill  them.  A  quick  and  rapid  eradication  is 
required  due  to  the  high  reproduction  rate  of  bed  bugs.  Only  PT18  products  are  intended  to 
control a bed bug infestation. It is crucial to exterminate bed bugs and prevent the infestation 
from  growing.  PT19  products  against  bed  bugs  are  not  intended  to  eradicate  a  bed  bug 
infestation. Repellent products against bed bugs can be useful as an accompanying measure in 
areas where the users  cannot exclude a bed bug infestation,  e.g. people sleeping in hotels  or 
hostels. In such rooms, the repellent products could be used to protect themselves as well as 
their luggage. 

A  sign  of  bed  bug  presence  includes  bites  on  the  exposed  skin  (small  red  itchy  bumps).  Bed 
bugs usually bite at night while their hosts are asleep. If observed, confined locations such as 
mattress linings or  furniture gaps  should be inspected  for faecal spotting and the presence  of 
bed bugs. 

5.6.5.3.1.1 Biology 

Bed bugs belong to the order of Hemiptera, Family Cimicidae. Adults body sizes range between 
4.5 mm and 8.5 mm. 

 
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Bed bug nymphs, adults and their eggs are found in cracks and crevices in the surroundings of 
the hosts’ sleeping area, e.g. furniture joints, along linings of mattresses, behind paintings and 
in  the  seams  of  furnishings.  Bed  bugs  have  a  strong  aggregation  behaviour  and  leave  their 
hiding  place  only  to  feed.  They  are  negatively  phototactic,  nocturnal  and  are  rarely  seen 
outside the harbourage, except in cases of heavy infestations. 

Female bed bugs can lay up to 150 eggs during their lifetime. Depending on the frequency of 
blood meals, temperature and humidity, bed bugs can have a lifetime expectancy of more than 
a  year.  They  are  able  to  survive  for  months  without  feeding.  The  nymphs  hatch  from  small 
white  eggs  after  7-10  days  at  room  temperature  and  earlier  at  higher  temperatures.  Each  of 
the  5  nymphal  stages  needs  a  blood  meal  to  complete  development  to  the  next  instar.  The 
whole life-cycle from egg to adult takes a minimum of 7 weeks. Blood meals are obligatory for 
each development stage, egg and sperm production. 

5.6.5.3.2 Dossier requirements  

Dossier requirements are stated in the Introduction (see section 5.6.5.1.3). 

5.6.5.3.2.1 Test species  

A  product  intended  for  use  as  a  repellent  or  attractant  against  bed  bugs  should  be  tested 
against the common bed bug (C. lectularius). If the tropical bed bug C. hemipterus is claimed, 
this species should be tested. The origin of the strain has to be documented in the test report, 
as  well  as  the  number  of  generations  for  which  the  bed  bugs  have  been  reared  in  the 
laboratory (for details see Introduction, section 5.6.5.1.3). 

Testing of products that target bed bugs should be conducted with adult and nymph bed bugs. 
Bed bugs should be tested not less than seven days after the last blood meal. 

Due to the specificity of certain active substances, e.g. pheromones, for products based on an 
active  substance  with  a  species-specific  mode  of  action,  only  effects  against  bed  bug  species 
that  have  been  tested  under  simulated-use  and/or  field  conditions,  depending  on  the  type  of 
claim, should be claimed on the product label and in the SPC.  

5.6.5.3.2.2 Requirements per type of claim and test methods 

These products will mostly not be able to eliminate an existing infestation and are only useful 
as  a  preventive  measure.  Unless  otherwise  proven  in  efficacy  trials,  the  label  and  the  SPC 
should  include  a  wording  like:  “Repellents/attractants  should  only  be  used  as  a  preventive 
measure, e.g. to prevent the spreading of bed bugs via infested luggage”. 

A laboratory test is not required for product authorisation but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided.  

5.6.5.3.2.2.1 Repellent products intended for use as surface treatment 

For  product  authorisation  purposes  the  efficacy  of  repellent  products  should  be  proven  in  a 
simulated-use  test  according  to  the  instructions  for  use  (test  design  example  see 
5.6.5.3.2.2.1.1). 

Test duration  should be according to the label  claim. If the product is  intended to protect  the 
user  while  in  a  bed  or  during  sleeping/overnight  a  minimum  efficacy  time  of  8  hours  is 
necessary to cover the natural bed bug activity over night. 

The product should be applied according to the use instructions and the repellent width should 
be recorded and stated on the label and in the SPC, e.g. “spray a band of at least 10 cm width 
around the area to be protected”. 

For products applied on surfaces two porous, e.g. plywood, carpet, fabric, and one non-porous 
surfaces,  e.g.  ceramic  tile,  painted  plywood  should  be  used  in  the  simulated-use  test,  for  a 
general label claim as “surface treatment”.  The efficacy of each surface should be proven  in a 
separate test, i.e. three tests for three different surfaces. For authorisation of a product to be 
used on a specific type of surface the efficacy for only this specific surface should be assessed. 
If a specific surface type is claimed, e.g. textiles, this surface has to be tested. 

 
 
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For  residual  efficacy,  bed  bugs  are  exposed  to  the  product  at  several  time  intervals  after 
application (including the end of the claimed period). 

If  outdoor  use  is  claimed,  the  treated  surface/substrate  or  repellent  product  and  control 
surfaces/substrates  have  to  be  kept  outside  to  ensure  adequate  weathering  and  the  outdoor 
conditions must be specified (for details see Introduction, section 5.6.5.1.4.1.3). 

Proposed  claims  regarding  the  performance  of  the  product  should  be  simulated  in  the  study. 
For  example,  for  the  claim  “unaffected  by  cleaning/vacuuming”,  the  surface  should  be 
repeatedly cleaned during the trial (for details see Introduction, section 5.6.5.1.3). 

The repellents used for the test should be identical to the product to be marketed. 

5.6.5.3.2.2.1.1 Simulated-use test for repellents for surface treatment  

Mandatory requirements: 

•  Tests  should  be  conducted  with  at  least  20  bed  bugs  (10  nymphs  and  5  adult  females 

and 5 adult males) per replicate. 

•  Replicates:  A  minimum  of  5  independent  replicates  should  be  performed  (each 

treatment and negative control). 

•  Environmental conditions must be specified for the test itself and during the storage of 
treated  substrates/surfaces  or  repellent  products  (temperature,  humidity, 
the 
photoperiod,  ventilation).  The  room  temperature  should  be  kept  at  22°C±4°C,  with  a 
relative  humidity  of  30-70%.  When  efficacy  under  tropical  conditions  is  claimed,  test 
parameters  should  be  adapted  accordingly.  If  the  product  is  intended  to  be  used  in 
ventilated rooms the simulated-use test should be executed in a room with ventilation. 

•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8), with bed bugs tested within the first hour. 

For  products  which  act  as  contact  repellents  or  at  a  short  distance  with  a  limited  release  into 
the  air,  one  of  the  following  set-up  examples  could  be  used:  an  open  arena  or  a  three-
chambers-system,  see  Appendix  18,  Wang  C.,  et  al.,  or  Vander  Pan,  A.,  et  al.  respectively. 
Both test examples determine if a bed bug is repelled by treated surfaces in the presence of a 
human  host  mimic  (CO2  and  a  heat  source).  However,  for  a  claim  “prevents  biting”,  testing 
needs  to  be  conducted  with  human  volunteers  in  order  to  determine  the  CPT.  Other  test 
designs than the following examples can be accepted if the protocol is scientifically valid. 

Simulated-use arena set-up 

Test design: In the centre of the test arena (at least 0.6 m2) a small bed or an imitation with 
four legs, e.g. a chair or small table is placed. Onto the simulated bed, a CO2 source (minimum 
release rate: 100 ml/min) and additionally a heat source (optional) should be placed to mimic a 
human host. Under each leg, a bed bug interceptor, i.e. a double-walled bed bug trap, where 
the insects are being trapped between both walls, which form a ring around the bed leg should 
be placed as a collection vessel. The repellent product should be applied on the outer walls of 
the  four  interceptors  at  the  recommended  rate(s)  following  the  product  use  instructions.  In 
case  that  the  test  surface  is  larger  than  the  interceptors  are  able  to  represent,  then  the 
interceptors can be placed on treated surfaces. Therefore, different surfaces can be treated and 
placed directly under the legs of the simulated bed. Bed bugs are placed in a harbourage, e.g. 
a pocket made of paper towel and tape easily opened with scissors in the centre of the arena 
right under the bed. After acclimatisation (at least 1 hour), the harbourage should be opened. 
The  test  arena  should  be  lined  with  material,  e.g.  paper  and  masking  tape  which  enables 
normal bed bug movements. Inner walls of the arena should be treated with a substance which 
prevents bed bugs from escaping. 

The same test design can be used for the evaluation of products claimed to protect goods e.g. 
when applied  on surfaces like  suitcases to prevent bed bug spreading. Instead of a  simulated 
bed, e.g. a suitcase filled with worn clothing or something similar is placed in the centre of the 
test arena. The repellent product should be applied according to the label claim and SPC, e.g. 
on the suitcase surface, or as a barrier around the item that is to be protected. An interceptor 
should be used to collect the bed bugs that cross the border treated  with the repellent in the 
case  of  a  barrier  treatment.  A  harbourage  for  the  bed  bugs  should  be  placed  in  one  of  the 

 
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corners  of  the  test  arena.  After  acclimatisation  (at  least  1  hour),  the  harbourage  should  be 
opened. 

Proof  of  non-insecticidal  efficacy:  To  proof  whether  insecticidal  effects  could  be  caused  by 
contact/exposure to the test repellent product bed bugs tested within the first hour should be 
used. Mortality of these insects should be monitored 24 hours after the end of the test. 

Efficacy assessment: The repellent effect of the product is determined by comparing the results 
obtained in the treated replicates with the ones from the control replicates. Different methods 
of recording the efficacy of the products can be used in the test design, e.g. sticky traps,  video 
camera, etc.). 

Simulated-use three-chambers-system 

Test design: The test system consists of three closed chambers joined with connector tubes. In 
the  first  chamber  (harbourage  chamber),  a  sealed  harbourage  with  the  test  organisms  is 
provided,  e.g.  a  pocket  made  of  paper  towel  and  tape  easily  opened  with  scissors.  After  a 
minimum  of  1  h  of  acclimatisation  the  harbourage  should  be  opened.  In  the  middle  chamber 
(test chamber) the treated surface is placed. The test chamber is connected to a third chamber 
(host  chamber)  containing  a  CO2  source  and  a  heat  source.  The  connector  tube  between  the 
test  and  host  chamber  should  protrude  into  the  host  chamber.  A  collecting  vessel  should  be 
placed  under  the  open  end  of  the  connecting  tube.  The  collecting  vessel  should  contain  filter 
paper  as  a  harbourage  and  the  inner  walls  should  be  treated  with  a  substance  that  prevents 
bed bugs from escaping from the vessel.  

Efficacy can be evaluated by counting the number of bed bugs which have crossed the surface 
in  the  test  chamber.  The  assessment  should  be  based  on  the  counting  of  the  bed  bugs  that 
cross  the  test  chamber  regardless  of  whether  they  are  collected  in  the  vessel  or  still  in  the 
tube. If bed bugs are found in the treated chamber or on the treated surface, they should be 
considered as non-repelled. 

To mimic the human host’s CO2 emission, the amount of CO2 should be adjusted according to 
the  size  of  the  chambers  and  the  distance  between  each  other.  Saturation  with  CO 2  can  be 
avoided by using a suction pump reaching in the harbourage chamber. The heat source inside 
the  test  chamber  should  be  adjusted  to  a  temperature  of  37°C±2°C.  The  connector  tubes 
should be lined with material, e.g. masking tape or paper which is not slippery for bed bugs. 

Proof  of  non-insecticidal  efficacy:  To  proof  whether  insecticidal  effects  could  be  caused  by 
contact/exposure  to  the  test  repellent  product  bed  bugs  tested  within  in  the  first  hour  should 
be used. Mortality of these insects should be monitored 24 hours after the end of the test. 

Efficacy assessment: The repellent effect of the product is determined by comparing the results 
obtained in the treated replicates with the ones from the control replicates. Different methods 
of recording the efficacy of the products could be used in the test design, e.g. stick traps, video 
camera etc. 

5.6.5.3.2.2.2 Products intended for use as topical repellents for human skin 

For product authorisation purposes the efficacy of repellent products should be proven in: 

•  a  simulated-use  test  according  to  the  instruction  for  use  (test  design  example  see 

5.6.5.3.2.2.2.1), or 

•  a field trial according to the instruction for use (specifications see 5.6.5.3.2.2.2.2). 

The repellents used for the test should be identical to the product to be marketed. 

Test duration should be according to the claim. If the product is intended to protect while in a 
bed or during sleeping/overnight a minimum efficacy time of 8 hours is necessary to cover the 
natural bed bug activity over night. 

5.6.5.3.2.2.2.1 Simulated-use test 

Mandatory requirements: 

•  Volunteers: Efficacy data of at least 10 different volunteers (preferably  volunteers with 
different  hairiness  of  the  arms,  different  genders,  age:  18  -  65  years)  should  be 
collected  since  repellence/attractiveness  to  bed  bugs  varies  considerably  between 

 
 
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human  individuals.  For  CPT  calculation  valid  data  of  at  least  10  different  volunteers 
must be used. 

Volunteers  need  to  be  fully  informed  about  the  aim,  the  procedure,  and  the  expected 
duration  of  a  study.  The  study  should  be  carried  out  in  compliance  with  the  national 
ethics  regulation.  They  also  need  to  be  informed  about  potential  side  effects  such  as 
allergic skin reactions caused by the product. Every side effect observed during the test 
should  be  mentioned  in  the  test  report.  Their  participation  is  voluntary  and  can  be 
recalled  at  any  time  before  and  during  the  study.  12  hours  before  and  during  testing, 
volunteers  should  avoid  nicotine,  alcohol,  fragrances  (perfumes,  body  lotions,  soap, 
etc.) and repellent products. Prior to the application of the repellent, the skin is washed 
with fragrance-free soap and rinsed with water. 

•  Tests should be conducted with at least 4 adult bed bugs per hour per volunteer. 

•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8), with bed bugs tested within the first hour. 

A  possible  test  set-up  is  given  below;  other  test  designs  than  the  following  example  can  be 
accepted if the protocol is scientifically valid.  

Test  design:  The  repellent  product  is  applied  to  human  forearms  or  lower  legs.  As  a  negative 
control, an untreated arm or leg of the same test person will be tested. This also serves to pre-
screen  bed  bugs  (adults)  for  sufficient  crawling  activity.  Bed  bugs  (adults)  showing  sufficient 
crawling activity are used for the test on the treated arm immediately after the control test. 

Bed  bugs  (adults)  must  be  active  for  host  searching  behaviour.  Therefore,  tests  should  be 
performed  under  red  dim  light  during  the  bed  bugs’  scotophase  (dark  phase)  and  after  a 
starvation period of a minimum of 1 week after the last blood meal. Bed bugs can be put on a 
piece  of  cardboard  or  other  material  which  is  held  right  above  the  treated  skin.  Then  it  is 
observed whether the bed bugs crawl down onto the skin. 

Exposure  periods  should  take  place  every  30  or  60  minutes  with  the  first  being  30  minutes 
after the product application. 2 to 5 adult bed bugs should be tested on each volunteer during 
each test period, giving a total of at least 4 bed bugs per hour per volunteer. 

A  bed  bug  that  does  not  move  at  all  on  the  test  arm,  even  after  moving  on  the  control  arm, 
has to be excluded from the test, and another bed bug should be used instead. Every bed bug 
should only be used once for a test to avoid habituation effects. When a bed bug starts to bite 
during the test, this bed bug should be removed directly from the skin. When a bed bug starts 
to  bite  hereby  on  the  treated  area  this  bed  bug  is  considered  as  non-repelled.  Biting  can  be 
prevented by constant observation. 

Efficacy  assessment:  While  testing  for  repellence,  an  endpoint  for  failure  of  repellence  for 
subjects treated with the recommended label dose should be selected. Efficacy failure in a test 
to determine CPT is the time from application of a repellent until efficacy failure by a confirmed 
event (definition see Introduction, section 5.6.5.1.5). Repellent efficacy should be based on the 
median or mean CPT. 

Proof of non-insecticidal efficacy: Bed bugs tested within the first hour (at least 50 individuals, 
with 5 individuals per volunteer) are used to proof the non-insecticidal efficacy. The bed bugs 
should  be  kept  separately  in  the  laboratory  and  mortality  should  be  recorded  24  hours  after 
exposure. 

5.6.5.3.2.2.2.2 Field trials 

If  the  field  trials  are  conducted,  they  must  take  place  in  highly  infested  buildings  with  an 
appropriate bed bug density. At least 30 adult bed bugs should be found within the room upon 
visual inspection. If no bed bugs are visible, the bed bug infestation should be determined by 
trapping  with  a  CO2  and  heat  source.  At  least  30  adults  should  be  trapped  within  12  hours. 
These  tests  should  preferably  take  place  in  Europe  or  other  relevant  regions  according  to  the 
claims, e.g. tropical regions.  

At least 10 volunteers (preferably an equal number of males and females; age: 18 – 65 years) 
should  be  included  in  the  field  trial,  and  the  same  number  in  the  negative  control  group  (see 
also 5.6.5.3.2.2.2.1 volunteers).  

 
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Volunteers must be protected from bed bug bites. This can be achieved by closely monitoring 
bed bug activity, e.g. under red light, or the use of interceptors. 

5.6.5.3.2.2.3 Repellents applied on clothing 

For product authorisation purposes the efficacy of repellent products should be proven in: 

•  a  simulated-use  test  according  to  the  instruction  for  use  (test  design  example  see 

5.6.5.3.2.2.3.1), or 

•  a field trial according to the instruction for use (specifications see 5.6.5.3.2.2.2.2). 

Test duration should be according to the claim. If the product is intended to protect while in a 
bed or during sleeping/overnight a minimum efficacy time of 8 hours is necessary to cover the 
natural bed bug activity over night. 

For  residual  efficacy,  bed  bugs  are  exposed  to  the  product  at  several  time  intervals  after 
application (including the end of the claimed period). 

Proposed claims regarding the protection of a specific type of fabric must be simulated, i.e. the 
same type of fabric has to be used in the simulated-use test. 

Proposed  claims  regarding  the  performance  of  the  product  need  to  be  simulated  in  the  study 
(for details see Introduction, section 5.6.5.1.3.5).  

For  specific  claims,  e.g.  prevention  of  bites  through/prevention  of  bites  next  to  the  treated 
clothes, relevant tests have to be submitted. 

The repellents used for the test should be identical to the product to be marketed. 

5.6.5.3.2.2.3.1 Simulated-use test  

For clothing, a test set-up similar to the test described in 5.6.5.3.2.2.2.1 would be suitable; a 
negative control should be included. Efficacy data of at least 10 different volunteers (preferably 
volunteers  with  different  genders;  age:  18  -  65  years)  should  be  collected  since 
repellence/attractiveness to bed bugs varies considerably between human individuals. For CPT 
calculation  valid  data  of  at  least  10  different  volunteers  must  be  used.  Tests  should  be 
conducted with at least 5 adult bed bugs per hour per volunteer. 

For other textiles or fabrics a simulated-use test similar to the three-chamber-system could be 
used  (see  5.6.5.3.2.2.1.1).  A  minimum  of  5  independent  replicates  each  with  at  least  10  bed 
bugs  should  be  performed  (each  treatment  and  negative  control).  Justify,  whether  individual 
bed bugs or groups are tested corresponding to the claim. 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  surfaces  (temperature,  humidity  and  photoperiod,  ventilation).  The  room  temperature 
should be kept at 22°C±4°C with a relevant humidity of 30-70%. When efficacy under tropical 
conditions is claimed, test parameters should be adapted accordingly. 

Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see  Introduction, 
section 5.6.5.1.3.8), with bed bugs tested within the first hour. 

5.6.5.3.2.2.4 Repellent products intended for use as spatial repellents  

Spatial  repellents  can  for  example  be  used  together  with  PT18  products,  to  make  bed  bugs 
leave  their  hiding  places  and  come  in  contact  with  the  insecticide,  see  Appendix  18:  Benoit, 
J.B. et al.. 

For product authorisation purposes the  efficacy of spatial  repellent products  should be proven 
in a simulated-use test according to the instruction for use. 

The repellents used for the test should be identical to the product to be marketed, and the test 
procedure should reflect the claim. 

When  the  claim  states  that  the  product  should  be  used  in  ventilated  rooms  the  opening  of 
windows and doors should be simulated in the test. 

 The  room  size  stated  in  the  SPC  and  on  the  label  must  be  used  in  the  efficacy  trial.  For  the 
extrapolation to larger room sizes than proven in the efficacy test a justification is necessary. 

 
 
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For a general claim “spatial repellents” the repellent effect must be proven. For a specific claim 
“dispelling”,  the  dispelling  effect  must  be  proven.  If  a  claim  states  that  the  product  prevents 
insects from entering a space, a simulated-use test proving the entry reduction is necessary. 

If  a  claim  states  that  the  product  prevents  insects  from  biting/probing,  a  simulated-use  test 
proving the biting/probing inhibition with a volunteer in the room is necessary. 

In  case  of  a  specific  claim  regarding  the  use  in  specific  climate  conditions,  e.g.  high 
temperature or tropical conditions the efficacy must be proven under these conditions. 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  substrates/surfaces  or  repellent  product  (temperature,  humidity,  photoperiod, 
ventilation). 

Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see  Introduction, 
section 5.6.5.1.3.8). 

5.6.5.3.2.2.5 Attractants without PT18 active substances  

For  product  authorisation  purposes  the  efficacy  of  attractants  in  traps  should  be  proven  in  a 
simulated-use test according to the instruction for use (see 5.6.5.3.2.2.5.1)  

The attractants, e.g. the trap, used for the test should be as similar as possible to the product 
to be marketed (for details see Introduction, section 5.6.5.1.3.7).  

5.6.5.3.2.2.5.1 Simulated-use test 

The test should simulate the claimed use of the product. Depending on its use pattern, whether 
the  product  is  intended  to  attract  bed  bugs  towards  a  harbourage  or  “pull”  them  away  from 
human  hosts,  the  product  performance  of  an  attractant  product  may  be  determined  e.g.  by 
comparing  its  effect  on  bed  bugs  to  that  of  bed  bug  aggregation  cues  or  host  cues  such  as 
carbon  dioxide  and  body  heat.  Therefore,  in  the  experiment  either  host  cues  such  as  carbon 
dioxide  and  body  heat  or  bed  bug  aggregation  cues  deposited  in  a  harbourage  should  be 
presented  as  an  alternative  to  the  attractant  formulation  in  the  same  arena.  Observations  of 
bed  bug  location  should  be  recorded  at  the  end  of  the  exposure  period.  The  exposure  period 
should  be  according  to  the  SPC  and  label  claim.  Testing  in  the  dark  under  red  light  is 
recommended. 

Replicates: A minimum of 5 independent replicates each with at least 20 bed bugs (10 nymphs, 
5  adult  females,  5  adult  males)  should  be  performed  (each  treatment  and  negative  control). 
Justify, if individual bed bugs are tested instead of groups. 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  substrates/surfaces  or  attractant  product  (temperature,  humidity,  photoperiod, 
ventilation).  Room  temperature  should  be  kept  at  22°C±4°C,  with  a  relative  humidity  of  30-
70%.  When  efficacy  under  tropical  conditions  is  claimed,  test  parameters  should  be  adapted 
accordingly. 

Any bycatch of non-target insects has to be recorded and identified at least to order, preferably 
to the family level. 

5.6.5.3.2.2.6 Attractants in PT18 bait products 

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for  bait 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods). 

5.6.5.3.3 Assessment of authorisation  

5.6.5.3.3.1 Norms and criteria  

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for bed bugs in the following way: 

Repellents against bed bugs: 

Non-insecticidal  efficacy  has  to  be  proven  in  a  simulated-use  test  if  it  cannot  be  waived  (for 
details see Introduction, section 5.6.5.1.3.8). 

Test duration should be according to the claim. If the product is intended to protect while in a 
bed or during sleeping/overnight a minimum efficacy time of 8 hours is necessary to cover the 
natural bed bug activity over night. 

 
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For a claim “prevents bites” or “prevents the  spreading  of bed bugs”,  e.g. for luggage, 100% 
repellency are required and the CPT needs to be proven. 

For repellent products intended for use as surface or spatial treatment: 

The required results for laboratory test, simulated-use tests or field tests are: 

•  ≥ 80% repellency within the test period (or according to the claim), from the beginning 

and until the end of the claimed efficacy period. 

For  products  intended  for  use  as  topical  repellents  for  human  skin  and  repellents  applied  on 
clothing: 

The required results for laboratory test, simulated-use tests or field tests are: 

•  during the claimed protection period complete protection should be proven expressed as 

CPT (for details see Introduction, section 5.6.5.1.5). 

Attractants without PT18 active substances  

The required results in the laboratory test or the simulated-use test are: 

•  at least a ratio of 4:1 of  bed bugs trapped in the trap with attractant  compared to the 
control  trap  within  the  test  period  (or  according  to  the  claim),  from  the  beginning  and 
until the end of the claimed efficacy period;  

•  ≥  80%  of  the  test  individuals  trapped  within  the  test  period  compared  to  the  negative 
control (or according to the claim), from the beginning and until the end of the claimed 
efficacy period 

Test duration should be according to the claim. If the product is intended to protect while in a 
bed or during sleeping/overnight a minimum efficacy time of 8 hours is necessary to cover the 
natural bed bug activity over night. 

5.6.5.4 Biting midges (Culicoides, veterinary)  

5.6.5.4.1 Introduction 

Biting  midges  (Diptera,  Ceratopogonidae,  Culicoides  Latreille)  are  well  known  veterinary 
disease  vectors  and  nuisance  in  livestock.  They  transmit  a  wide  range  of  filaria  including 
Onchocerca  cervicalis  in  Europe.  A  range  of  viruses  has  been  isolated  from  biting  midges 
including  African  horse  sickness  virus  (AHSV),  a  highly  infectious  disease  that  may  causes 
serious  lung  congestion  and  may  lead  to  the  death  of  horses.  This  disease  is  mainly  reported 
from  Spain  and  Portugal.  Also,  the  closely  related  epizootic  haemorrhagic  virus  and  the 
Bluetongue virus (BTV) are transmitted by biting midges. BTV affects ruminants such as deer, 
cattle,  sheep  and  goat  and  is  transmitted  by  species  like  Culicoides  imicola,  Culicoides 
obsoletus,  and  others.  Apart  from  Culicoides  biting  midges  role  as  vectors  they  can  also 
represent  a  serious  nuisance.  Horses  may  suffer  from  equine  summer  eczema  (sweet  itch) 
caused by an allergic reaction to saliva from biting midges. The mane, back and tail areas are 
often  affected,  but  it  can  be  seen  all  over  the  body.  Ponies  and  islandic  horses  are  more 
sensitive  to  sweet  itch  than  other  horses.  Cattle  may  suffer  from  the  mere  numbers  of  biting 
midges attacking in evenings with no wind, and high temperature and humidity in areas close 
to the biting midges breeding areas. 

5.6.5.4.1.1 Biology 

Culicoides biting midges are the smallest members of the blood-feeding Diptera with sizes of 1-
4 mm. They have broiled or spotted wings which characteristically are found directed backward 
when at rest. Culicoides biting midges are recorded from all over the world apart from the most 
extreme Arctic and Antarctic regions. There are over 1400  Culicoides species in the world and 
at  least  83  species  are  described  from  Europe.  Biting  midges  feed  blood  on  a  relatively  wide 
range of mammals and birds. Only the females are blood-suckers. The activity of the different 
species  varies  through  the  season.  Some  have  a  single  annual  activity  optimum,  others  have 
two  while  some  are  present  throughout  the  season.  The  periods  of  abundance  have  great 
importance  for  those  species  that  are  vectors  for  pathogens.  The  female  midges  must  have 
blood meals to develop eggs and they need  new blood meals for each of their ovarian cycles. 
This  means  that  a  female  must  seek  and  find  a  new  host  several  times  in  her  lifetime.  The 
Culicoides biting midges fly mainly in the dusk but sometimes also early in the morning. In rare 
cases,  they  attack  in  the  middle  of  day  if  the  conditions  are  suitable  i.e.  low  wind,  high 

 
 
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temperature and high humidity. Various  Culicoides species are to some extent host-specific in 
their blood-feeding, some favour mammals, some birds and some amphibians. Many species of 
Culicoides  show  a  different  preference  for  blood-feeding  on  individual  host  animals;  some 
favour to feed the belly and udder region on cattle, while other species prefer the extremities, 
the back, or the head. 

5.6.5.4.2 Dossier requirements 

Dossier requirements are stated in the Introduction (see section 5.6.5.1.3). 

5.6.5.4.2.1 Test species  

As of now, only two biting midge species, i.e.  C. sonorensis and C. nubeculosus can be raised 
in  the  laboratory,  and  probably  none  of  them  can  act  biologically  relevant  in  captivity  when 
sensitive biological parameters such as host-seeking is investigated. The important issue, when 
the  efficacy  of  a  repellent  is  measured,  is  whether  a  product,  when  applied  to  the  host  in  a 
relevant environment, can overrule all the key stimuli that attract biting midges to their host. 
Tests  with  laboratory  colonies  of  relevant  Culicoides  species  as  well  as  with  field  collected 
specimens are applicable provided such testing is justified in detail. 

•  Products  intended  for  use  as  topical  or  spatial  repellent  should  be  tested  with  the  claimed 

host, e.g. cattle, horse, sheep. 

•  Products intended as a general Culicoides biting midge repellent; in case of a general claim 
against biting midges, repellent testing should be performed with biting midges present on 
the  hosts  in  field  trials  or  simulated-use  test.  Specimens  can  be  collected  during  the  trial 
with aspirators on the control animals for later identification in the laboratory. Alternatively, 
species from the study area are identified previous to the study. 

• 

If a specific species is claimed, then efficacy against this species must be demonstrated.  

•  Products  intended  for  use  as  attractants  should  be  tested  against  claimed  Culicoides 

species. 

•  Test  organisms  should  belong  to  species  encountered  in  Europe  or  European  territories 
(overseas  territories).  In  addition,  efficacy  against  other  organisms  not  encountered  in 
Europe or European territories should be demonstrated  on the relevant species if  claimed. 
The  climate  conditions  (temperature,  humidity)  should  be  representative  to  European 
conditions. 

•  When  use  in  tropical  areas  is  claimed,  test  at  temperatures  >  30°C  should  be  provided. 
Additionally,  it  should  be  specified  against  which  Culicoides  biting  midges  the  product  is 
effective, and these should be tested. 

• 

In  case  of  specific  claims,  e.g.  effective  at  high  temperature,  in  contact  with  water,  host 
sweating, etc., efficacy must be demonstrated in the relevant situations. 

5.6.5.4.2.2 Requirements per type of claim and test methods 

A laboratory test is not required for product authorisation but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/  attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided. 

5.6.5.4.2.2.1 Products intended for use as repellents 

The efficacy of a product should be shown in either a robust simulated-use test, where animals 
can be tethered and observed close by or a field test where the animals are loose/untethered 
and  observed  from  a  distance  to  establish  the  efficacy  and  persistence  of  the  product  when 
used according to the label claim in a real-life situation.  

For PT19 products which are applied on animals, no common protocols are available. However, 
the  European  Medicines  Agency  (EMA)  has  published  several  guidelines  for  the  evaluation  of 
ectoparasiticides, see Appendix 18. These guidelines may be adapted for the demonstration of 
the efficacy of repellent products to be applied on animals, but any well documented protocol 
can be used, provided it is scientifically sound.  

 
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It is important to give a detailed description of the circumstances in which the experiments are 
conducted, with special emphasis on the ratio of treated to untreated host animals at each trial 
location and trial replicate. It may be of consequence for the trial outcome whether all hosts in 
a herd are treated, compared to a situation where only a minor part of the hosts in a herd is 
treated.  It  is  also  of  importance  that  the  diurnal  rhythm  of  biting  midge  activity  is  taken  into 
consideration in the setup of the efficacy trials and reported in detail. If the product is part of a 
push-pull strategy, e.g. hosts with repellent combined with attracting traps, then such a setup 
must be reported in detail as well. 

If treated horse rugs or similar covers are claimed to be effective in the field, then it must be 
demonstrated  effective  in  the  field.  It  is  not  important  to  make  an  evaluation  of  the  biting 
midges on the  rug itself, but the emphasis must be on the exposed parts of  the host such as 
the neck, head, legs and belly, including the sheath on males and udder on females, which are 
thin-skinned areas and very attractive to several blood-feeding species.  

Tests must be presented with all host animal species claimed. Bridging  efficacy data from one 
host  species  to  another  can  be  accepted  based  on  robust  scientific  justification  taking  into 
account e.g. the difference in dosing between species (a small goat vs. a large cow), whether 
sweating is possible or not. 

5.6.5.4.2.2.1.1 Laboratory test 

Laboratory tests of repellents are not advised, as the differences in food seeking biology of the 
relevant species are huge. It would be possible to do laboratory tests with a few biting midge 
species  as  they  can  be  kept  in  the  laboratory,  and  therefore  specific  claims  on  these  species 
may  be  backed  by  data  generated  in  laboratory  experiments,  but  it  will  not  be  possible  to 
extrapolate such data to cover any other species encountered on grazing livestock. 

5.6.5.4.2.2.1.2 Simulated-use test 

In a simulated-use test different parameters are controlled. A simulated-use test of a repellent 
with full control of host animals and absence of their normal avoidance behaviour can be used 
to evaluate the efficacy and the persistency of the treatment; topically, spatial or impregnated 
textiles. The product should be applied according to the claim. Environmental conditions must 
be  specified  at  the  beginning  and  during  the  test  (temperature,  humidity,  photoperiod).  No 
specific temperature or humidity are recommended, but the environmental conditions must be 
suitable  to  have  biting  midge  activity  high  enough  to  challenge  the  product.  A  control 
treatment  without  repellent  should  be  included  in  all  trials  to  secure  a  measure  of  Culicoides 
biting midge activity.  

A simulated-use test must be conducted at a time in the season, as well as on days where the 
relevant  Culicoides  species  are  active  in  the  field.  Depending  on  the  target  animal  species, 
several factors, e.g. hair length, thickness of the coat, grooming, etc. might impact the efficacy 
of  topical  repellents.  Explain  in  the  test  report/documentation  the  circumstances  around  the 
choosing  of  the  test  animals.  If  animals  of  different  colour  are  chosen  be  sure  not  to  have  a 
majority of light coloured animals. Ideally, light coloured animals are not included, but state on 
the  label  if  the  product  is  for  a  specific  colour  of  animals  if  only  light  coloured  animals  were 
tested a sound justification is needed to include all fur colours in the claim. For products only 
tested  on  e.g.  pied  (black  or  red)  cattle,  it  can  be  necessary  to  state  on  the  label  that  the 
product is for pied (black  or red) cattle. For products tested on  sheep, it can be necessary to 
test  on  both  sheared  and  unsheared  sheep,  a  sound  justification  is  needed  if  only  sheared  or 
unsheared sheep are tested. 

At least two groups of host animals must be used, one group treated with the product and one 
group  with  no  treatment.  At  least  ten  different  individuals  are  suggested  to  be  used  in  each 
group. In the case of horses, a setup could be 2x5 horses with a treatment switch after e.g. a 
week,  depended  on  the  residual  efficacy  of  the  tested  product,  tested  at  one  location,  or  2x5 
animals  tested  at  two  different  locations.  At  the  end  of  the  test  period,  there  must  be  2x10 
valid datasets. Each treatment group should be housed in separate enclosures throughout the 
course of the trials to prevent cross infestation or cross-contamination. 

Preferably, no test animals should have a recent history of treatment with insecticide/acaricide 
or  antiparasitic  agents.  Concerning  endo-antiparasitic  agents  this  is  acceptable,  but  details 
(product,  dose,  application  rate,  application  method,  date  of  the  last  treatment,  and  residual 
efficacy) must then be given in the report; the controls and the treated groups must have the 

 
 
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same status. A safety margin for externally applied anthelmintics is the end of last application 
+1 month. The history of the treated and the control groups should be comparable. 

Animals should be treated with the test product once the infestation has become  established. 
The  infestation  should  be  documented  by  e.g.  recordings  of  the  numbers  of  biting  midges  or 
photographs.  As  mentioned  above  specific  Culicoides  species  tend  to  prefer  specific  areas  on 
the hosts. Therefore, collections or counts must be conducted in the same area on all hosts i.e. 
on the back,  or on the  belly  etc. It is acceptable that counts/collections are done  on  tethered 
hosts. If the product is acting at very close range it may be possible to make the evaluation by 
treating parts of the host, like one treated side of the belly compared to the equivalent area on 
the other, untreated, side  of the belly. The advantage  of  such a setup is the neutralisation  of 
individual  host  differences  in  biting  midge-attraction.  Collection  can  be  done  using  an 
electrically driven aspirator in a defined time period. The attractiveness of the test animals or 
experimental groups should be tested prior to treatment. 

Percentage  inhibition  (landing)  is  based  on  the  number  of  biting  midges  landing  on  the 
untreated control animals and the number of biting midges landing on the treated animals: 

Pct inhibition: ((LandingControl – LandingTreated)/LandingControl) x 100. 

Alternatively  or  in  addition  to  midge  landing  catches,  also  the  frequency  of  certain  horse’s 
avoidance behaviour according to Mottet et al. (2018), see Appendix 18, can be recorded as a 
substitute, provided the abundance of the target species predominantly causing the avoidance 
behaviour  is  measured  at  the  beginning  and  the  end  of  the  trial.  Test  designs  may  also  be 
adapted from Japin, M. and Haanen, G. A. Y.(2013), see Appendix 18. 

5.6.5.4.2.2.1.3 Field trials 

In  a  field  trial  the  animals  must  have  the  opportunity  to  display  normal  avoidance  behaviour 
considering how the product is intended to be used. The tests may be conducted as described 
for  the  simulated-use  test  above,  with  the  adjustment  that  there  is  no  interference  with  the 
host animal’s normal behaviour in the field.  

The field trials should be conducted on two different geographical locations.  

At least two groups of host animals must be used, one group treated with the product and one 
group  with  no  treatment.  At  least  10  different  individuals  are  suggested  to  be  used  in  each 
group. Due to feasibility and cost-effectiveness, it is possible to use 10 animals in one week (5 
each  in  the  treatment  and  control  group)  and  then  use  the  same  individuals  again  in  the 
second week, but swap treatments. Each animal would then act as its own control. Any residual 
activity  of  the  product  has  to  be  excluded.  The  field  trials  can  be  performed  twice  with  5 
animals (in each of the control and treatment groups) each time at the same location, or with 5 
animals  (in  each  of  the  control  and  treatment  groups)  at  two  different  locations.  At  least  10 
animals (each as control and treatment) must be tested. The two locations can be in the same 
country or region, e.g. two villages. In the case of cattle, the test can be performed at only one 
location using 10 cows for the control and 10 cows for the treatment. 

As  documentation  for  persistency,  counts  are  repeated  with  chosen  intervals  of  lengths 
depending on the claim. If there is a claim for use on wet/sweating hosts, then data must be 
provided  showing  that  the  product  is  effective  under  such  circumstances.  The  guideline  from 
the  EMA:  “Guideline  on  specific  efficacy  requirements  for  ectoparasiticides  in  cattle”,  and 
Herholz et al. (2016), see Appendix 18, may be of inspiration for field trial setup. 

5.6.5.4.2.2.2 Attractants without PT18 active substances 

Use of an attractant in  a trap in  a push-pull setup may be suggested. A trap could be loaded 
with  CO2,  heat,  octenol,  butyric  acid,  etc.  In  such  a  setup  the  biting  midge  load  will  be 
measured as described above. 

For  product  authorisation  purposes,  the  efficacy  of  an  attractant  in  a  trap  without  an  active 
substance according to PT18 should be proven in a simulated use test or a field test according 
to the instructions for use. 

The attractants, e.g. the traps, used for the test should be as similar as possible to the product 
to be marketed (see Introduction section 5.6.5.1.3.7) and the trap should be designed in a way 
that  reduces  bycatch  to  an  absolute  minimum.  Traps  should  be  tested  on  their  own,  with  a 
control tested separately in a similar test setting. 

 
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5.6.5.4.2.2.3 Attractants in PT18 bait products  

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods). 

5.6.5.4.3 Assessment of authorisation 

5.6.5.4.3.1 Norms and criteria 

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for biting midges on livestock in the following way: 

Products intended for use as repellent on animals (including a delivery system) 

•  A  simulated  use  test  or  a  field  test  demonstrating  ≥  80%  repellency  within  the  test 
period (according to the claim), directly after product application and at the end of the 
claimed use period; 

•  All  claimed  target  host  animals  must  be  tested.  Claims  on  herds  as  well  as  individuals 

are acceptable and should be tested as such; 

• 

In  case  of  specific  claims,  e.g.  effective  at  high  temperature,  in  contact  with  water, 
when  the  host  are  sweating,  when  horses  are  at  work  etc.  these  should  be 
demonstrated. 

Products  intended  for  use  as  a  spatial  repellent  or  for  use  as  attractants  without  PT18  active 
substances 

A trap with an added attractant should catch significantly more biting midges than one without 
the attractant. The traps used for the test should be as similar as possible to the product to be 
marketed.  

•  A simulated use test or a field test demonstrating ≥ 80 % efficacy (i.e. 80% of the test 
individuals trapped) within the test period according to the claim, directly after product 
application and at the end of the claimed use period; 

•  All claimed target species must be tested; 

•  At least a ratio of 4:1 of test individuals trapped in the trap with attractant compared to 
the  control  trap  within  the  test  period  (or  according  to  the  claim),  from  the  beginning 
and until the end of the claimed efficacy period. 

The  product  label  and  SPC  should  state  that  the  entire  insect  population  might  not  be 
caught/repelled  by  this  type  of  product;  the  label  claim  should  be  “reduces”  and  not 
“protects”/”protection”, with no mention of a CPT.  

5.6.5.5 Cockroaches 

5.6.5.5.1 Introduction  

Cockroaches are a common and persistent problem in many households, commercial premises, 
domestic  buildings,  private  and  public  areas,  restaurants,  industry.  These  crawling  insects 
(although several species with the ability to fly) are scavengers, allowing them to readily adapt 
to  changing  food  availability.  Cockroaches  may  act  as  a  mechanical  vector  of  various 
pathogens,  e.g.  can  carry  bacteria  such  as  Salmonella  in  areas  co-inhabited  by  humans. 
Cockroaches  are  also  identified  as  a  major  cause  of  allergies  and  asthma,  particularly  in 
children. Amongst the crawling insects, cockroaches are one of the most difficult to control. 

5.6.5.5.1.1 Biology  

Cockroaches belong to the (sub-) order Blattodea. There are over 3500 species of cockroaches, 
but  only  a  few  are  considered  domestic  pests  in  the  EU.  The  German  cockroach,  Blattella 
germanica,  the  Oriental  cockroach,  Blatta  orientalis,  and  the  American  cockroach,  Periplaneta 
americana, are the most common. 

Upon hatching from an egg capsule, cockroaches begin their nymphal stage and moult through 
various  instars  until  reaching  the  adult  stage.  The  nymphal  stages  are  smaller  than  the  adult 
stage without fully developed wings and sex reproduction organs. The time of development can 
take weeks or months depending on the species and the surrounding environmental conditions. 
Depending  on  the  temperature,  the  eggs  of  the  German  cockroach  hatch  after  3  to  5  weeks, 

 
 
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the nymphal development with 5 to 7 moultings takes 40 days to 6 months and the adults live 
for about 6 months. 

In temperate European countries,  most cockroach species  will almost  never be  found outside, 
with foraging activities almost entirely within human-made structures. 

5.6.5.5.2 Dossier requirements  

Dossier requirements are stated in the Introduction (see section 5.6.5.1.3). 

5.6.5.5.2.1 Test species  

For  a  general  claim  “against  cockroaches”,  two  key  species  should  be  tested:  one  small 
species, belonging to the family Ectobiidae, the German cockroach B. germanica, and one large 
species,  belonging  to  the  family  Blattidae,  either  the  Oriental  cockroach  B.  orientalis  or  the 
American  cockroach  P.  americana.  For  a  specific  claim,  testing  against  the  claimed  species  is 
required. 

Due to the specificity of certain active substances, e.g. pheromones, for products based on an 
active substance with a species-specific mode of action, only effects against cockroach species 
that  have  been  tested  under  simulated-use  and/or  field  conditions,  depending  on  the  type  of 
claim, can be claimed on the product label and in the SPC. 

For  insects  from  laboratory  rearing  used  in  the  efficacy  studies,  age  and  feeding  condition 
should be reported (for details see Introduction, section 5.6.5.1.3). 

5.6.5.5.2.2 Requirements per type of claim and test methods 

A laboratory test is not required for product authorisation but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided. 

5.6.5.5.2.2.1 Repellent products intended for use as surface treatment 

For product authorisation purposes the efficacy of repellent products should be proven in: 

•  a  simulated-use  test  according  to  the  instruction  for  use  (test  design  example  see 

5.6.5.5.2.2.1.1), or 

•  a  field  trial  according  to  the  instruction  for  use  (test  design  example  see 
5.6.5.5.2.2.1.2)  and  additionally  a  laboratory  trial  testing  the  required  different 
surfaces. 

A  simulated-use  test  testing  the  product  and  the  negative  control  in  one  set-up  can  only  be 
used  as  additional,  supportive  information.  Testing  of  the  product  and  the  negative  control 
separately is necessary. 

Products applied onto surfaces may act either by evaporation or on the surface itself. 

For products applied on surfaces, two porous and one non-porous surface should be used, e.g. 
ceramic  tile,  plywood,  painted  plywood,  stainless  steel,  concrete,  for  a  general  claim  as 
“surface treatment”. The efficacy of each surface should be proven in a separate test, i.e. three 
tests for three different surfaces. For authorisation of a product to be used on a specific type of 
surface the efficacy for only this specific surface should be assessed. 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  substrates/surfaces  or  repellent  product  (temperature,  humidity,  photoperiod, 
ventilation). The temperature would be expected to fall in the range of 19–29°C. When efficacy 
at high temperatures is claimed, 40°C should be the test temperature. 

For  residual  efficacy,  cockroaches  are  exposed  to  the  product  at  several  time  intervals  after 
application (including the end of the claimed period). 

If  outdoor  use  is  claimed,  the  treated  surface/substrate  or  repellent  product  and  control 
surfaces/substrates  have  to  be  kept  outside  to  ensure  adequate  weathering  and  the  outdoor 
conditions  must  be  specified  (for  detailed  information  regarding  the  requirements  see 
Introduction, section 5.6.5.1.4.1.3). 

 
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Proposed  claims  regarding  the  performance  of  the  product  should  be  simulated  in  the  study. 
For  example,  for  the  claim  “unaffected  by  cleaning/vacuuming”,  the  surface  should  be 
repeatedly cleaned during the trial (for details see Introduction, section 5.6.5.1.3).  

The  repellents  used  for  the  test  should  be  identical  to  those  used  in  the  product  to  be 
marketed. 

5.6.5.5.2.2.1.1 Simulated-use test 

Mandatory requirements: 

• 

In the simulated-use test that evaluates the  repellent  efficacy of products intended for 
use  as  surface  treatment  the  insects  must  have  a  choice  to  be  in  contact  with  the 
repellent or not Product and negative control are separately tested. 

•  Conducted in arenas with a surface of at least 0.5 m2 (intended as whole surface if the 

test is performed connecting 2 arenas).  

•  The test arena should be equipped with water, food and a shelter. The positions of food 
and  water  in  the  arena  must  ensure  that  the  cockroaches  are  forced  to  come  into 
contact with the product.  

•  Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative 
control)  should  be  performed.  10  adult  males,  10  adult  females  and  20  nymphs  are 
included in each replicate. 

•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8). 

•  The observation of cockroaches should be conducted at different time points depending 

on the claim (e.g. after some hours, 1 day and up to 7 days post-exposure).  

•  Product application should be done outside of the test room. 

Different  methods  of  monitoring  the  cockroaches  and  assessing  the  repellent  effect  of  the 
product  are  possible,  e.g.  by  using  a  photo  or  video  observation  system  or  by  applying 
adhesive material to the floor of the arena.  

The  adhesiveness  of  adhesive  material  varies  and  depends  on  the  cockroach  species,  and  it 
must  be  excluded  that  cockroaches  are  repelled  by  this  material.  Therefore,  the  catching 
efficacy  of  the  adhesive  material  must  be  demonstrated  for  each  cockroach  species.  At  least 
80% of cockroaches should be caught by the adhesive material. This can be demonstrated e.g. 
by catching of at least 80% of the test individuals in the control. 

Proof of non-insecticidal effects: The mortality of the cockroaches can be monitored at the end 
of  each  trial,  if  no  adhesive  material  is  used.  Alternatively,  the  same  test  set-up  can  be  used 
without adhesive material to monitor the mortality of the cockroaches at the time points used 
in the repellent efficacy tests (day(s) post exposure). 

Efficacy assessment: The potential repellent effect of the product is  determined by  comparing 
the number of cockroaches on the area  protected with the repellent product to the number of 
cockroaches on the same area in the negative control.  

5.6.5.5.2.2.1.2 Field trial 

In  the  field  trials  the  product  is  tested  in  an  actual  use  situation,  for  instance  in  an  infested 
home or warehouse and applied according to the directions for use on the label and in the SPC 
and against the claimed species (see 5.6.5.5.2.1). An example of an appropriate study design, 
e.g. number of replicates, observation parameters for a field trial in an infested area including 
a description of a pre-test to determine the initial population size can be found in Appendix 19. 

Non-insecticidal  efficacy,  if  it  cannot  be  waived  (for  details  see  Introduction,  section 
5.6.5.1.3.8) should be examined at the end of the trial, if possible. In the case it is not possible 
mortality has to be evaluated in a simulated use test. 

5.6.5.5.2.2.2 Products intended for use as spatial repellents 

For product authorisation purposes the efficacy of spatial repellents should be proven in: 

•  a  simulated-use  test  according  to  the  instructions  of  use  (test  design  example  see 

5.6.5.5.2.2.2.1), or 

 
 
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•  a 

field  trial  according  to  the 

instruction 

for  use  (test  design  example  see 

5.6.5.5.2.2.2.2). 

Only products that affect the target organisms by application of the product in the air and not 
by  walking  on  treated  surfaces  can  be  claimed  as  spatial  repellents  (for  more  details  see 
Introduction, section 5.6.5.1.4). 

The repellents used for the test should be identical to the product to be marketed. 

 The  room  size  stated  in  the  SPC  and  on  the  label  must  be  used  in  the  efficacy  trial.  For  the 
extrapolation to larger room sizes than proven in the efficacy test a justification is necessary. 

For  a  general  claim  “spatial  repellents”  the  repellent  effect  must  be  proven.  For  the  specific 
claim “dispelling” the dispelling effect must be proven. 

5.6.5.5.2.2.2.1 Simulated-use test 

Mandatory requirements: 

•  The simulated-use test that evaluates the repellent efficacy of products intended for use 
as spatial repellents has to be performed in test chambers with a volume adapted to the 
claim stated in the SPC (at least 20 m3).  

•  For ambient repellents for protection of large rooms, e.g. diffusers to protect rooms with 
a defined volume, a double room test should be performed, with at least one 20 m³ test 
room  for  product  application.  Such  spatial  repellent  products  can  repel  cockroaches  to 
protect rooms from entering and/or dispel cockroaches from already infested areas. 

•  Both test rooms/boxes contain water, food and a shelter.  

•  Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative 
control)  should  be  performed.  Each  replicate  has  to  use  10  adult  males,  10  adult 
females and 20 nymphs. 

•  Product  and  negative  control  are  separately  tested.  The  negative  control  should  be 

conducted with the same set-up as the treatment without the spatial repellent. 

•  Environmental  conditions  must  be  specified  for  the  test  itself  (temperature,  humidity, 
photoperiod, ventilation). The temperature would be expected to fall in the range of 19–
29°C.  When  efficacy  at  high  temperatures  is  claimed,  40°C  should  be  the  test 
temperature.  

•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8).  

The  following  test  design  example  could  be  used  for  observing  both  effects  (repelling  and 
dispelling).  Other  test  designs  than  the  following  example  can  be  accepted  if  the  protocol  is 
scientifically valid. 

General set-up 

The repellent product is applied in a test room of at least 20 m³, which is adjacent to a second 
room of the same or different volume. In the test room, a box (at least 0.5 m²) is placed in the 
corner furthest away from the repellent product. This box is connected on the shorter side via a 
tube  (passage)  with  a  second  box  (at  least  0.5  m²)  in  the  adjacent  room.  The  inner  walls  of 
both boxes are covered with talcum to prevent cockroaches from escaping. A shelter is placed 
in  both  boxes  opposite  the  passage  entry.  Alternatively,  the  test  can  be  conducted  in  two 
connected test rooms in which the cockroaches can freely move.  

For  recording,  of  the  cockroaches  at  certain  times  photo  or  video  observation  systems  above 
both boxes could be used to avoid disturbing the insects.  

After  product  activation  and  opening  of  the  passage,  the  light  is  turned  off  and  the  system 
should  be  exposed  to  a  twilight/darkness  rhythm  of  about  12  hours,  undisturbed  over  the 
entire  test  period  (only  for  evaluation  purposes  the  experimental  chamber  should  be  entered 
with low lighting). 

Repellent effect 

The repellent product is applied and activated according to the instructions for use in the test 
room. In each replicate, a group of cockroaches is introduced in the box in the untreated room. 

 
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During the acclimatisation period of at least 2 hours the entry of the passage tube is blocked. 
Afterward  the  passage  is  opened,  and  the  cockroaches  can  move  freely  between  both 
boxes/rooms. 

Dispelling effect 

In each replicate, a group of cockroaches is introduced in the box in the test room. During the 
acclimatisation  period  of  at  least  2  hours,  the  entry  of  the  passage  tube  is  blocked.  After 
acclimatisation the dispelling product is applied and activated according to the instructions for 
use  in  the  test  room  and  the  entry  is  opened.  During  the  test  period,  the  cockroaches  can 
move freely between both boxes/rooms. 

Evaluation for both test set-ups (repellent or dispelling effect) 

The  number  of  cockroaches  in  each  box/room  should  be  recorded  at  different  time  points 
depending  on  the  claim,  e.g.  after  some  hours,  1  day  or  up  to  7  days  post-exposure. 
Cockroaches are  exposed to the test set-up at several time intervals  after product application 
including the end of the claimed residual period. The number of cockroaches in each box/room 
should  be  recorded  for  all  time  intervals  at  the  same  time  post-exposure,  e.g.  1-day  post-
exposure. 

Efficacy assessment: The potential repellent effect of the product is determined by  comparing 
the  number  of  cockroaches  in  the  box/room  with  the  repellent  product  to  the  number  of 
cockroaches in the room outside the product application. 

In a control test without product application side effects of the test rooms should be excluded 
and unimpeded passage through the rooms must be demonstrated for each cockroach species 
that  is  observed  in  the  test  system.  In  the  control  test,  the  cockroaches  must  be  equally 
distributed in both test  rooms/boxes, as evidenced by non-significant  differences between the 
two rooms/boxes.  

5.6.5.5.2.2.2.2 Field trial 

In  the  field  trials  the  product  is  tested  in  an  actual  use  situation,  for  instance  in  an  infested 
home or warehouse and applied according to the directions for use on the label and in the SPC. 
An example of an appropriate study design, e.g. number of replicates, observation parameters 
for a field trial in an infested area including a description of a pre-test to determine the initial 
population size can be found in Appendix 19. 

Proof  of  non-insecticidal  effects,  if  it  cannot  be  waived  (for  details  see  Introduction,  section 
5.6.5.1.3.8) should be examined at the end of the trial, if possible. In the case it is not possible 
mortality has to be evaluated in a simulated use test. 

5.6.5.5.2.2.3 Attractants without PT18 active substances 

For product authorisation purposes the efficacy of attractants in traps should be proven in: 

•  a simulated-use test according to the instruction for use, or  

•  a field test according to the instruction for use. 

Any attractant should be tested against a negative control. 

The attractants, e.g. the traps, used for the test should be as similar as possible to the product 
to  be  marketed  (for  details  see  Introduction,  section  5.6.5.1.3.7).  Any  bycatch  of  non-target 
insects has to be recorded and identified at least to order, preferably to the family level. 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  substrates/surfaces  or  attractant  product  (temperature,  humidity,  photoperiod, 
ventilation). The temperature would be expected to fall in the range of 19–29°C. When efficacy 
at high temperatures is claimed, 40°C should be the test temperature.  

If  outdoor  use  is  claimed,  the  test  should  be  performed  outdoors  and  the  attractant  product 
and control have to be kept outside to ensure adequate weathering and the outdoor conditions 
must be specified (for details see Introduction, section 5.6.5.1.4.1.3). 

Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative  control) 
should be performed. For a simulated-use test each replicate has to include 10 adult males, 10 
adult females and 20 nymphs. 

 
 
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5.6.5.5.2.2.4 Attractants in PT18 bait products 

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for  bait 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods). 

5.6.5.5.3 Assessment of authorisation  

5.6.5.5.3.1 Norms and criteria  

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for cockroaches in the following way: 

Products intended for use as repellent products as surface treatment or spatial treatment 

Non-insecticidal  efficacy  has  to  be  proven  in  a  simulated-use  test  if  it  cannot  be  waived  (for 
details see Introduction, section 5.6.5.1.3.8). 

The required results for the different tests are: 

Laboratory test or simulated-use test: 

•  ≥  80%  repellence  or/and  dispellence  (depending  on  the  claim)  compared  to  the 
negative control is demonstrated within the test period (or according to the claim), from 
the beginning and until the end of the claimed efficacy period 

Field trial: 

•  ≥  80%  repellence  or/and  dispellence  relative  to  either  control  sites  or  pre-treatment 
levels  within  the  test  period  (or  according  to  the  claim),  from  the  beginning  and  until 
the end of the claimed efficacy period 

Products intended for use as attractants without PT18 active substances 

The required results for laboratory test, simulated-use tests, or field tests are: 

• 

at least a ratio of 4:1 of test individuals trapped in the trap with attractant compared to 
the  control  trap  within  the  test  period  (or  according  to  the  claim),  from  the  beginning 
and until the end of the claimed efficacy period;  ≥ 80% of the test individuals trapped 
within the test period compared to the negative control (or according to the claim), from 
the  beginning  and  until  the  end  of  the  claimed  efficacy  period  against  the  claimed 
species. 

5.6.5.6 Fleas 

5.6.5.6.1 Introduction  

Fleas  are  small  flightless  insects  that  survive  as  external  or  epidermic  parasites  of  mammals 
and birds. They are vectors of different zoonotic diseases, such as plague, murine typhus, cat 
scratch fever and allergic dermatitis triggered by flea saliva also with implications for humans. 
The cat flea Ctenocephalides felis is the most important ectoparasite of domestic cats and dogs 
worldwide.  

The  Oriental  rat  flea  Xenopsylla  cheopis,  also  known  as  the  tropical  rat  flea,  is  a  parasite  of 
rodents  and  humans,  and  is  a  primary  vector  for  bubonic  plague  and  murine  typhus.  The 
human flea Pulex irritans is a cosmopolitan flea species that has a wide host spectrum. It can 
also  be  an  intermediate  host  for  the  flea  tapeworm  cestode  Dipylidium  caninum.  Tunga 
penetrans (also known as chigoe flea or jigger) is a parasite of mammals (dogs and humans) in 
most tropical and sub-tropical climates causing an inflammatory skin disease (tungiasis). 

5.6.5.6.1.1 Biology  

Of the over 2000 species of fleas (Siphonaptera), the cat flea C. felis and the dog flea C. canis 
are the most common in-home pests in the EU. 

Fleas undergo complete metamorphosis (egg, larva, pupa, adult) and the lifecycle begins when 
an adult female finds a suitable host. Once found, the female flea remains on this host for the 
rest  of  its  life.  Females  produce  several  eggs  after  each  blood  meal  and  can  produce  several 
hundred eggs in their lifetime. The laid eggs fall off the animal host and develop in the areas 
where the host spends its time. The eggs tend to accumulate in the lowest areas such as deep 
in  fibres  of  carpets,  in  cracks  and  crevices  in  the  floor,  furniture,  and  furnishings  or  behind 
mouldings. 

 
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Larvae require high protein food for their survival. By feeding  on the dry faeces of adult fleas, 
larvae  receive  the  protein-rich  food.  The  adult  flea  takes  in  more  blood  from  the  host  than 
necessary for nourishment and excretes the remaining blood in almost pure form. Once dried, 
the  faeces  fall  off  the  host  where  the  larvae  can  feed.  The  larvae  spin  a  cocoon  including  the 
surrounding material and begin the pupal state. 

During  the  pupal  stage,  the  development  can  be  delayed  if  the  environmental  conditions  are 
unfavourable.  The  pupae  can  enter  into  the  rest  of  several  months  (diapause).  An  adult  flea 
emerges  from  the  pupae  after  stimulation  by  external  cues  that  indicate  a  host  in  the  close 
surrounding. After emergence, a flea must usually find a host (located using visual and thermal 
cues) within a week, or it risks death due to desiccation.  

Complete  development  from  egg  to  adult  occurs  in  as  little  as  two  weeks  but  can  take  much 
longer depending on environmental conditions. 

The  appearance  and  parasitic  behaviour  of  Tunga  penetrans  differ:  it  is  the  smallest  known 
flea, measuring 1 mm. After a bloodmeal, males are still mobile like other fleas, but the female 
flea  burrows  head-first  into  the  host's  skin,  leaving  the  caudal  tip  of  its  abdomen  visible 
through  an  orifice  in  a  skin  lesion.  This  orifice  allows  the  flea  to  breathe,  defecate,  mate  and 
expel eggs while feeding from blood vessels. The flea’s abdomen swells with eggs later in the 
cycle, reaching a size up to 1 cm. 

5.6.5.6.2 Dossier requirements  

General dossier requirements are stated in the Introduction (see section 5.6.5.1.3). 

5.6.5.6.2.1 Test species  

For a general claim “against fleas”, the product should normally be tested on adult cat fleas C. 
felis  or  adult  dog  fleas  C.  canis.  If  products  are  exclusively  claimed  against  larvae  than  the 
efficacy  should  be  demonstrated  for  that  developmental  stage  instead  of  adults.  For  specific 
species  claims,  tests  should  be  performed  with  the  claimed  flea  species.  Test  species  should 
also be specific or known to be an ectoparasite of the host to be protected, e.g.  C. canis for a 
product used for dogs, notably in case of topical products. 

Due to the specificity of certain active substances, e.g. pheromones, for products based on an 
active  substance  with  a  species-specific  mode  of  action,  only  effects  against  flea  species  that 
have been tested under simulated-use and/or field conditions, depending on the type of claim, 
should be claimed on the product label and in the SPC. 

For  insects  from  laboratory  rearing  used  in  the  efficacy  studies,  age  and  feeding  conditions 
should be reported (for more details see Introduction, section 5.6.5.1.3). 

5.6.5.6.2.2 Requirements per type of claim and test methods 

A laboratory test is not required for product authorisation but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided.  

5.6.5.6.2.2.1 Products intended for use as topical repellents for human skin or 
clothing 

For  product  authorisation  purposes  the  efficacy  of  repellent  products  targeting  the  use  on 
human skin and clothing should be proven in: 

•  a  simulated-use  test  according  to  the  instructions  for  use  (test  design  example  see 

5.6.5.6.2.2.1.2), or 

•  a  field  trial  according  to  the  instructions  for  use  (test  design  example  see 

5.6.5.6.2.2.1.3). 

The repellent products used for the test should be identical to the product to be marketed. 

Proposed label claims regarding the performance of the product must be simulated (for details 
see Introduction, section 5.6.5.1.3.5).  

Proposed  label  claims  regarding  the  protection  of  a  specific  type  of  fabric  must  be  simulated, 
i.e. the same type of fabric has to be used in the simulated-use test. 

 
 
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5.6.5.6.2.2.1.1 Laboratory tests 

Mandatory requirements: 

• 

• 

• 

• 

A standardized laboratory in-vitro assay should be performed.  

Fabrics  or  clothing  should  be  treated  at  the  recommended  application  rate(s) 
according to the label claim and SPC. 

Replicates:  A  minimum  of  5  independent  replicates  (each  replicate  contains  10 
individually tested fleas) should be performed (each treatment and negative control). 

Environmental  conditions  must  be  specified  for  the  test  itself  and  the  drying  of  the 
product, and during storage of the treated fabrics or clothing (temperature, humidity 
and photoperiod). The temperature of the test room would be expected to fall in the 
range between 20-23°C. 

Other test designs than the following examples can be accepted if the protocol is scientifically 
valid. 

Warm Object Bioassay (see Appendix 18: Büchel K., Kleier S., Dautel H.) 

The  “Warm  Object  Bioassay”  is  suitable  for  the  determination  of  repellency  of  liquid 
formulations applied on fabrics/clothing and treated fabrics. The test should determine, if a flea 
is  repelled  by  treated  fabrics  in  the  presence  of  a  host  mimic  (heat  source).  The  experiment 
exploits  that  adult  fleas  are  attracted  to  warm  objects  corresponding  to  the  host  body 
temperature and are negatively geotactic. 

Test design: In the middle of a test arena (at least 900 cm²) a warm object is placed simulated 
by a narrow glass cylinder (high: > 20 cm) containing warm liquid (35–36°C) as heat source, 
so that the surface temperature of the cylinder  corresponds to the external body temperature 
of  the  host.  The  surface  of  the  cylinder  is  covered  by  filter  paper  or  test  fabric  (for  products 
applied on or incorporated in textiles). The  fabric used has to be representative  of the fabrics 
commercially used, e.g. thick uniform cotton for military clothing. The repellent product should 
be  applied  at  the  recommended  application  rate(s)  following  the  product  use  instructions  on 
the  test  fabric  or  clothing  and  must  be  dry  before  conducting  the  test.  Each  centimetre  a 
horizonal  line  is  drawn  on  the  fabric/filter  paper  to  determine  the  distance  movement  of  the 
fleas on the object as an additional behavioural parameter. 

In total at least 50 unfed adult fleas should be tested for each test product. After testing of 10 
fleas (= one replicate) the surface (test fabric/clothing) should be removed and replaced by a 
new  one  (newly  sprayed).  The  total  observation  time  for  each  individual  is  a  maximum  of  4 
minutes.  

A flea is not repelled when it, within the 4 minutes observation time, either  

•  climbs  or  jumps  onto  the  test  fabric/clothing  and  remains  on  the  treated  surface  for 

more than 2 minutes, or 

•  reaches the upper end of the warm object within 2 minutes. 

In all other cases, the fleas are considered as repelled. 

It  is  therefore  possible  to  test  contact  repellents  as  well  as  repellents  acting  over  short 
distance. 

Efficacy  assessment:  The  repellent  effect  is  evaluated  by  the  time  and  distance  fleas  stay  or 
crawl  on  the  “warm  object”.  Total  vertical  distance  movement  can  be  recorded  in  centimetre 
steps.  

Blood Feeding Assay 

The following test method is a suitable set-up for the determination of the repellency of liquid 
formulations. 

Test  design:  The  Blood  Feeding  Assay  consists  of  an  apparatus  used  basically  for  artificial 
feeding  for  adult  cat  fleas,  see  Appendix  18:  Wade,  S.E.,  Georgi,  J.  R..  The  automated, 
temperature-controlled feeding unit is covered with a membrane. The unit is subsequently filled 
with bovine blood and set at 37°C.  

 
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10 unfed adult fleas are transferred into a plexiglass ring (approximately 1.5 cm height and 4 
cm  diameter)  that  is  covered  with  nylon  gauze  on  both  sides.  Before  covering  the  plexiglass 
ring  with  the  fleas,  the  upper  side  of  the  nylon  gauze  is  treated  with  product  at  the 
recommended  application  rate(s)  following  the  product  use  instructions.  After  product 
application  and  the  completely  drying  of  the  nylon  gauze  the  feeding  unit  is  placed  on  the 
treated  nylon  gauze,  allowing  the  fleas  to  feed  through  the  gauze  and  the  membrane  for  1 
hour.  A  Petri  dish  with  filter  paper  is  placed  under  the  caged  fleas  to  evaluate  the  extent  of 
feeding by flea faeces or blood traces caused by accumulating on the filter paper in the course 
of feeding. 

Efficacy assessment: The repellent effect is determined by comparing the proportion of feeding 
between  treatment  and  control.  The  proportion  of  feeding  is  quantified  over  the  area  of  flea 
droppings  caused  through  blood-feeding  in  mm2  on  the  filter  paper  below  the  plexiglass  ring. 
Alternatively,  the  proportion  of  feeding  could  be  determined  by  evaluating  the  number  of 
engorged fleas. 

5.6.5.6.2.2.1.2 Simulated-use test 

Mandatory requirements: 

•  The  efficacy  of  repellent  products  applied  on  human  skin  or  clothes  according  to  the 
instructions for use should be proven in a simulated-use test. A test set-up similar to an 
“arm-in-cage”  or  “arm-to-cage”  test  would  be  suitable;  a  negative  control  should  be 
included. 

•  Efficacy  data  of  at  least  10  different  volunteers  (preferably  volunteers  with  different 
hairiness  of the arms,  different genders; age: 18 - 65 years) should  be collected since 
repellence/attractiveness  to  fleas  varies  considerably  between  human  individuals.  For 
efficacy assessment, valid data of at least 10 different volunteers must be used. 

•  Repellent products have to be applied at the recommended label dose. 

•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8). 

5.6.5.6.2.2.1.2.1 Volunteers 

Volunteers’ attractiveness is measured by exposing the untreated skin to caged populations of 
host-seeking,  pathogen-free,  unfed  fleas,  and  the  results  must  be  presented  in  the  report.  A 
minimum of 5 probings per minute must be achieved. To avoid unnecessary blood feeding, the 
volunteers  are  allowed  to  softly  brush  off  the  fleas  after  probing  with  a  soft  brush.  In  case 
probing activity is lower, a new cage with fresh fleas should be used or additional fleas could be 
added and reported.  

Volunteers need to be fully informed about the aim, the procedure and the expected duration 
of  a  study.  The  study  should  be  carried  out  in  compliance  with  the  national  ethics  regulation. 
They  also  need  to  be  informed  about  potential  side  effects  such  as  allergic  skin  reactions 
caused by the product. Their participation is voluntary and can be recalled at any time before 
and during the study, see Appendix 18: WHO and EPA guidelines. 12 hours before and during 
testing,  volunteers  should  avoid  nicotine,  alcohol,  fragrance  (perfumes,  body  lotions,  soap, 
etc.)  and repellent products. Any side effect  observed during the test  should be mentioned in 
the test report. 

Prior to the application of the repellent, the skin is washed with fragrance-free soap and rinsed 
with water. The hand should be covered with a glove that fleas cannot bite through during each 
exposure to the test insects. If the entire forearm is to be treated with repellent, the skin area 
should  be  estimated  according  to  recommendations  of  the  WHO:  the  mean  circumference  is 
calculated  by  measuring  the  circumference  at  the  wrist  and  elbow  and  multiplied  with  the 
length  of  the  arm  (between  wrist  and  elbow).  Alternatively,  an  "arm-to-cage"  test  can  be 
conducted,  i.e.  repellents  can  be  applied  to  a  defined  area  on  the  forearm,  e.g.  a  100  cm² 
window.  In  “arm-in-cage”  tests,  the  rest  of  the  arm  will  then  be  protected  from  bites  by  a 
custom-made  sleeve  (flexible  material  that  prevents  biting,  edges  are  not  treated  with  the 
product;  contamination  should  be  prevented).  The  use  of  a  defined  area  adds  to  a 
standardisation of test conditions; therefore, the use of a sleeve is recommended.  

 
 
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5.6.5.6.2.2.1.2.2 Efficacy testing 

Other test designs than the following examples can be accepted if the protocol is scientifically 
valid. 

A rectangular gauze cage (at least 27 L) with an opening for the arm on one side and fleas on 
the  other  side  in  a  given  harbourage  should  be  used.  For  acclimatization  fleas  should  be 
introduced in the cage 1 hour before the test. 

Prior  to  the  efficacy  test  of  the  treated  arm,  the  probing  pressure  (5  probings/minute)  of  the 
pathogen-free, unfed test fleas needs to be verified with the other, untreated control arm. The 
treated skin should be exposed in regular intervals, e.g. once an hour. 

Efficacy assessment:  While testing for repellence, an endpoint  for the failure  of  repellence for 
subjects treated with the recommended label dose should be selected. Efficacy failure in a test 
to determine CPT is the time from application of a repellent until efficacy failure by a confirmed 
event (definition see Introduction, section 5.6.5.1.5). Repellent efficacy should be based on the 
median or mean CPT (CPT calculation, see Introduction, section 5.6.5.1.5).  

Proof  of  non-insecticidal  efficacy:  After  the  test  fleas  should  be  kept  under  optimal  rearing 
condition to observe whether insecticidal effects are  seen after a contact/exposure to the test 
repellent product. Mortality of the insects should be monitored after 24 hours. 

5.6.5.6.2.2.1.3 Field trials 

Field tests allow the evaluation of topical repellents against wild flea populations under realistic 
conditions  at  the  recommended  label  dose.  In  contrast  to  simulated-use  tests  they  are, 
however,  subjected  to  greater  variation,  regarding  probing  activity,  flea  species,  population 
density, temperature, humidity, light conditions, etc. Probing pressure on untreated skin should 
be  at  least  five  fleas  within  one  minute.  Volunteers  (at  least  10;  preferably  volunteers  with 
different  hairiness  of  the  arms,  different  genders;  age:  18  -  65  years  -  for  more  details  see 
5.6.5.6.2.2.1.2.1)  trained  on  the  use  of  an  aspirator  can  serve  as  their  own  control  on  the 
other,  untreated,  arm  or  leg.  Alternatively,  10  untreated  volunteers  could  be  included  in  the 
field trial, as control. The forearm (between wrist and elbow) or lower leg (between knee and 
ankle)  serves  as  the  treatment  area.  The  skin  area  should  be  measured  and  treated  as 
described  under  5.6.5.6.2.2.1.2.1.  Product  application  must  follow  the  claimed  instruction  of 
use. The treated skin will be exposed in regular intervals. Frequency of assessments will be not 
less  than  every  hour  until  CPT  ends  (occurrence  of  the  first  confirmed  probing,  see 
Introduction, section 5.6.5.1.5). Exposure periods should be synchronized with the time when 
the  relevant  species  are  abundant.  The  treatment  of  arms  or  legs  should  also  be  consistent 
with the feeding preference of the target species. Time of day at which volunteers are treated 
and  at  which  exposure  started  and  ended  should  be  reported.  Fleas  that  probe  or  bite  during 
an  efficacy  test  can  be  collected,  where  possible,  for  later  identification.  Before  the  study  is 
conducted,  the  presence  of  the  relevant  flea  species  has  to  be  verified.  This  should  be 
documented in the study report. 

5.6.5.6.2.2.2 Products  intended  for  use  as  topical  repellents  on  animals  or  animal 
clothing 

For  product  authorisation  purposes  the  efficacy  of  repellent  products  targeting  the  use  on 
animal skin or animal clothing should be proven in: 

•  a  simulated-use  test  according  to  the  instructions  for  use  (test  design  example  see 

5.6.5.6.2.2.2.2), or 

•  a  field  trial  according  to  the  instructions  for  use  (test  design  example  see 

5.6.5.6.2.2.2.3). 

Proposed  label  claims  regarding  the  performance  of  the  product  should  be  simulated  in  the 
study.  For  the  claim  “unaffected  by  washing”,  the  label  and  the  SPC  must  indicate  how  often 
the animal can be washed without reducing the efficacy of the biocidal product. In the efficacy 
test,  the  test  individuals  should  be  repeatedly  washed  with  a  non-insecticidal,  fragrance-free 
shampoo during the trial according to the number of wash cycles indicated. If a product claim 
is  to  protect  the  entire  animal,  this  should  be  demonstrated  (notably  for  collars).  For  specific 
claims regarding products applied on clothes or fabrics see Introduction, section 5.6.5.1.3.5.  

 
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The  efficacy data should be  relevant to prove  the submitted  claims. Therefore, the  efficacy of 
biocidal  products  that  are  limited  to  a  body  area,  e.g.  collars  for  animals  must  be  proven  for 
the  whole  test  individual  that  should  be  protected.  If  only  specific  body  areas  intend  to  be 
protected, e.g. ears, udders, etc. then the efficacy for the claimed area must be proven. 

For specific claims (prevention of bites through/prevention of bites next to the treated clothes), 
relevant tests have to be submitted. 

5.6.5.6.2.2.2.1 Laboratory test  

A  standardized  laboratory  test,  e.g.  “Warm  Object  Bioassay”  (see  Appendix  18:  Büchel  K., 
Kleier  S.,  Dautel  H.)  can  be  used  to  observe  the  behavioural  response  of  fleas  to  a  treated 
surface (for a details see 5.6.5.6.2.2.1.1). 

5.6.5.6.2.2.2.2 Simulated-use test 

Repellents  for  use  on  animals,  e.g.  dogs,  cats  should  be  tested  in  a  simulated-use  test.  The 
repellent effect should be demonstrated for each target animal species claimed. Dogs should be 
used as a substitute for cats, as testing with cats can imply certain animal welfare issues. The 
repellent product is applied according to the instructions for use. If the product is acting at very 
close  range  it  may  be  possible  to  make  the  evaluation  by  treating  parts  of  the  host,  like  one 
treated side of the neck compared with the equivalent area on the other, untreated, side of the 
neck. The advantage of such a setup is the neutralisation of individual host differences in flea-
attraction.  The  control  body  part  should  resemble  the  treated  body  part,  e.g.  front-half  vs 
back-half or left side vs right side of the animal. In case of a restricted application, e.g. collar, 
a spot-on product with a claim of entire body protection, or for products acting in a wider range 
over the entire body the test should permit to validate the efficacy of this kind of application by 
testing the entire animal. In this case, untreated animals must be used as a negative control. 

Test animals: It is recommended to include at least 10 animals per treatment/control group of 
a different breed and sex, i.e. for testing on entire animals at least 10 treated and 10 negative 
control  individuals  are  necessary.  The  age,  hair/fur  length,  weight  and  coat  colour  of  each 
animal should be recorded. It is necessary that animals come from suitable, e.g. non-smoking 
households. Every side effect observed during the test should be mentioned in  the test report. 
24  hours  before  and  during  testing,  animals  should  not  be  treated  with  fragrance  (perfumes, 
soap,  etc.).  The  test  animal  must  be  free  of  fleas  and  should  not  be  protected  by  any 
repellent/insecticidal  residuals  caused  by  previous  repellent  and/or  insecticidal  treatment 
(veterinary  medicinal  product).  Therefore,  the  animals  shall  be  screened  and  cleaned  before 
the  repellent  is  applied.  Concerning  endo-antiparasitic  agents  this  is  acceptable,  but  details 
(product,  dose,  application  rate,  application  method,  date  of  the  last  treatment,  and  residual 
efficacy)  must  be  given  in  the  report  and  the  negative  controls  and  the  treated  groups  must 
have the same status. A safety margin for externally applied anthelmintics is 4 weeks after the 
end  of  the  last  application.  The  history  of  the  treated  and  the  control  groups  should  be 
comparable.  The  treatment  status  of  the  animals  should  be  specified  and  only  animals  for 
which the insecticidal/repellent effect can be excluded should be included in the study. 

Proof  of  non-insecticidal  efficacy,  if  it  cannot  be  waived  (for  details  see  Introduction,  section 
5.6.5.1.3.8):  After  the  test  fleas  are  maintained  in  a  test  system  to  observe  whether 
insecticidal effects are seen after exposure to the test repellent product. Mortality of the insects 
should be monitored after 24 hours. 

5.6.5.6.2.2.2.3 Field trials 

For PT19 products which are applied on animals, no common protocols are available. However, 
several guidelines for the evaluation of ectoparasiticides are available, please see Appendix 18. 
These guidelines can be adapted for the demonstration of the efficacy of repellent products to 
be  applied  on  animals.  If  field  trials  are  conducted,  they  must  take  place  in  an  area  with  an 
appropriate  flea  density.  These  tests  should  preferably  take  place  in  Europe.  Where  tests  in 
Europe are not possible, the conditions and flea species must be confirmed, and their relevance 
justified. 

The  repellent  effect  should  be  demonstrated  for  each  target  animal  species,  e.g.  dog,  horse, 
etc.  claimed.  Dogs  should  be  used  as  a  substitute  for  cats,  as  testing  with  cats  can  imply 
certain animal welfare issues.  

Test animals: For details see 5.6.5.6.2.2.2.2. 

 
 
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The investigator is reminded that the validity of the results is directly related to the degree of 
variability in the test. Increasing the number of test animals could increase the reliability of the 
test results, see Appendix 18: OPPTS 810.3300. 

The product should be applied according to the claim. 

Depending on the animal species, several factors, e.g. hair length, thickness of the coat, self-
grooming,  etc.  might  impact  the  efficacy.  These  factors  should  be  taken  into  account  in  the 
demonstration  of  the  efficacy.  The  two  groups  should  be  kept  separately,  to  avoid  contact  of 
the control animals with the treated fur. 

Claimed application rates should take into account the type and weight of the animals.  

Proof  of  non-insecticidal  efficacy,  if  it  cannot  be  waived  (for  details  see  Introduction,  section 
5.6.5.1.3.8):  During  the  trial  fleas  should  be  collected  and  after  the  trial  maintained  in  a  test 
system  to  observe  whether  insecticidal  effects  are  seen  after  exposure  to  the  test  repellent 
product. Mortality of the insects should be monitored after 24 hours. 

5.6.5.6.2.2.3 Repellent products intended for use as surface treatment  

For product authorisation purposes the efficacy of repellent products should be proven in: 

•  a  simulated-use  test  according  to  the  instruction  for  use  (test  design  example  see 

5.6.5.6.2.2.3.2), or 

•  a  field  trial  according  to  the  instruction  for  use  and  additionally  a  laboratory  trial  (test 

design examples see 5.6.5.6.2.2.3.1) testing the required different surfaces. 

Products applied onto surfaces may act either by evaporation or on the surface itself. 

For products applied on surfaces, two porous and one non-porous surface should be used, e.g. 
ceramic  tile,  plywood,  painted  plywood,  stainless  steel,  concrete,  for  a  general  label  claim  as 
“surface treatment”. The efficacy of each surface should be proven in a separate test, i.e. three 
tests for three different surfaces. For authorisation of a product to be used on a specific type of 
surface the efficacy for only this specific surface should be assessed. If a specific surface type 
is claimed, e.g. dog mattress, this surface has to be tested. 

The repellents used for the test should be identical to the product to be marketed. 

For residual efficacy, fleas are exposed to the product at several time intervals after application 
(including the end of the claimed period). 

Proposed  label  claims  regarding  the  performance  of  the  product  should  be  simulated  in  the 
study.  For  example,  for  the  claim  “unaffected  by  cleaning/vacuuming”,  the  surface  should  be 
repeatedly cleaned during the trial (for details see Introduction, section 5.6.5.1.3). 

5.6.5.6.2.2.3.1 Laboratory test 

A  laboratory  test  can  be  performed  to  observe  the  behavioural  response  of  fleas  to  treated 
surfaces,  e.g.  plywood,  carpet,  ceramic  tile,  painted  plywood,  stainless  steel,  concrete.  Other 
test designs than the following example can be accepted if the protocol is scientifically valid. 

Test design: A box (at least 120 cm²) covered with gauze for ventilation serves as test arena. 
The  relevant  surfaces  have  to  be  applied  with  the  repellent  product  at  the  recommended 
application rate(s) following the product use instructions. Half of the arena is covered with the 
treated and the other half with the untreated surface. An attractant source should be placed on 
the treated area. The negative control (e.g. entire arena covered with untreated surfaces) runs 
under the same conditions.  

10 unfed adult fleas per replicate are released on the untreated side of the arena. 

Replicates: A minimum of 5 independent  replicates should be performed (each treatment and 
negative control).  

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  substrates/surfaces  or  repellent  product  (temperature,  humidity,  photoperiod, 
ventilation).  Conditions  during  the  test  have  to  be  maintained  at  25°C±2°C  and  relative 
humidity  of  >  75%.  Conditions  during  the  storage  have  to  be  maintained  at  20-25°C  and  a 
natural humidity unless specific conditions are required according to the claim. 

 
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Efficacy  assessment:  The  number  of  fleas  on  the  treated  and  untreated  surface  should  be 
recorded.  The  distribution  of  the  fleas  in  the  control  treatment  replicates  has  to  be  non-
significantly different. 

5.6.5.6.2.2.3.2 Simulated-use test 

Mandatory requirements: 

•  Replicates:  A  minimum  of  5  independent  replicates  should  be  performed  (each 
treatment and negative control). Each replicate consists of at least 10 unfed adult fleas. 

•  Test conducted in an arena with a minimum size of 0.5 m2. 

•  Environmental conditions must be specified for the test itself and during the storage of 
the 
repellent  product  (temperature,  humidity, 
photoperiod,  ventilation).  The  temperature  would  be  expected  to  fall  in  the  range  of 
20-25°C. 

treated  substrates/surfaces  or 

•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8). 

•  For a claim “prevents biting”, testing needs to be conducted with volunteers, depending 
on the claim either humans or animals, in order to determine the CPT (CPT definitions 
and calculation see Introduction, section 5.6.5.1.5). For efficacy assessment, valid data 
of at least 10 different volunteers (humans or animals) must be used. 

The  following  choice-test  could  be  used  for  observing  the  repellent  effect  on  surfaces.  Other 
test designs than the following example can be used if the protocol is scientifically valid.  

Test  design:  In  a  ventilated  test  room  two  identical  types  of  surface,  e.g.  dog  basket  at  a 
distance  of  1  m  should  be  used  at  the  same  time.  The  repellent  product  is  applied  at  the 
recommended  application  rate(s)  following  the  product  use  instructions  on  one  surface, 
whereas  the  second  untreated  surface  represents  the  control.  For  easy  detection  of  fleas,  the 
surface  should  be  of  light  colour.  To  lure  the  free-ranging  fleas  to  the  surfaces  an  attractant 
source  and  optionally  a  heat  source,  e.g.  a  heating  mat,  are  used  to  simulate  the  body 
temperature of the host (35-37°C). 

10 unfed adult fleas mixed sexes per replicate are released in the middle of the test room equal 
distance  between  both  surfaces  and  can  move  freely  within  the  room.  Access  to  the  surfaces 
should be barrier-free and crawling under the surfaces should be inhibited. During the test-run, 
the  number  of  fleas  should  be  monitored  at  several  time  intervals  after  product  application 
(including the day of treatment and at the end of the claimed residual period) on both surfaces 
e.g. by high-resolution action cams installed above. 

Efficacy  assessment:  The  repellent  effect  (%)  should  be  calculated  relatively  to  the  negative 
control surface. The number of fleas on the treated and the negative control surface should be 
recorded. Individuals who are neither on the test surface nor on the control surface should be 
not included in the evaluation.  

5.6.5.6.2.2.4 Repellent products intended for use as spatial repellents 

For  product  authorisation  purposes  the  efficacy  of  spatial  repellents  should  be  proven  in  a 
simulated-use test according to the instructions for use.  

The repellents used for the test should be identical to the product to be marketed. 

When the label claim says that the product should be used in ventilated rooms the opening of 
windows and doors should be simulated in the test. 

The  room  size  stated  in  the  SPC  and  on  the  label  must  be  used  in  the  efficacy  trial.  For  the 
extrapolation to larger room sizes than proven in the efficacy test a justification is necessary. 

For a general claim “spatial repellents” the repellent effect must be proven. For a specific claim 
“dispelling” the dispelling effect must be proven. 

If  a  claim  states  that  the  product  prevents  insects  from  biting/probing,  a  simulated-use  test 
proving  the  biting/probing  inhibition  with  a  volunteer  in  the  room,  depending  on  the  claim 
either humans or animals, is necessary. 

 
 
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In  case  of  a  specific  claim  regarding  the  use  in  specific  climate  conditions,  e.g.  high 
temperature or tropical conditions the efficacy must be proven under these conditions. 

Replicates: A minimum of 5 independent  replicates should be performed (each treatment and 
negative control). 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  substrates/surfaces  or  repellent  product  (temperature,  humidity,  photoperiod, 
ventilation). 

Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see  Introduction, 
section 5.6.5.1.3.8). 

5.6.5.6.2.2.5 Attractants without PT18 active substances  

For product authorisation purposes the efficacy of attractants in traps should be proven in: 

•  a simulated-use test according to the instructions for use, or 

•  a field test according to the instructions for use. 

Any attractant should be tested against an untreated control. 

The attractants, e.g. the traps, used for the test should be as similar as possible to the product 
to  be  marketed  (for  details  see  Introduction,  section  5.6.5.1.3.7).  Any  bycatch  of  non-target 
insects has to be recorded and identified at least to order, preferably to the family level. 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  substrates/surfaces  or  attractant  product  (temperature,  humidity,  photoperiod, 
ventilation). The temperature would be expected to fall in the range of 20-25°C. 

If  outdoor  use  is  claimed,  the  test  should  be  performed  outdoors  and  the  attractant  product 
and control have to be kept outside to ensure adequate weathering and the outdoor conditions 
must  be  specified  (for  detailed  information  regarding  the  requirements  see  Introduction, 
section 5.6.5.1.4.1.3). 

Replicates: A minimum of  5 independent  replicates should be performed (each treatment and 
negative control). 

5.6.5.6.2.2.6 Attractants in PT18 bait products  

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for  bait 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods). 

5.6.5.6.3 Assessment of authorisation  

5.6.5.6.3.1 Norms and criteria 

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for fleas in the following way: 

Products intended for use as repellent for human or animal skin, clothing:  

Non-insecticidal  efficacy  has  to  be  proven  in  a  simulated-use  test  if  it  cannot  be  waived  (for 
details see Introduction, section 5.6.5.1.3.8). 

The required results for the different tests are: 

Laboratory test (not required for product authorisation): 

•  100% repellency within the test period (or according to the claim), from the beginning 

and until the end of the claimed efficacy period. 

Simulated-use test and field trial: 

•  during the claimed protection period complete protection should be proven expressed as 

mean or median CPT (for details see Introduction, section 5.6.5.1.5). 

Products intended for use as general surface and spatial repellents:  

Possible  insecticidal  effects  have  to  be  examined  in  a  simulated-use  test  (for  details  see 
Introduction, section 5.6.5.1.3.8). 

 
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The  required  results  for  laboratory  test  (not  required  for  product  authorisation  of  spatial 
repellents), simulated-use test and field trials are: 

•  ≥ 80% repellency within the test period (or according to the claim), from the beginning 

and until the end of the claimed efficacy period. 

If “prevention of bites” is claimed, then 100% repellency (CPT) is also required for surface and 
spatial repellents for the claimed period. 

Products intended for use as attractants without PT18 active substances: 

The required results in the simulated-use test are: 

•  at least a ratio of 4:1 of test individuals trapped in the trap with attractant compared to 
the control trap within the test period (or according to the claim), from the beginning 
and until the end of the claimed efficacy period;  

•  ≥  80%  of  the  test  individuals  trapped  within  the  test  period  compared  to  the  negative 
control within the test  period (or according to  the claim), from the beginning and until 
the end of the claimed efficacy period. 

5.6.5.7 Flies on grazing cattle, horses and other livestock 

5.6.5.7.1 Introduction 

The  fly-fauna  on  cattle,  horses  and  other  livestock  is  complex  and  many  of  the  species  have 
different  impacts  on  their  hosts.  Flies  on  grazing  livestock  in  pastures  represent  a  very 
significant  problem.  These  flies  are  divided  into  two  groups,  the  biting  flies  and  the  sucking 
flies. Biting flies are penetrating the skin of the host to suck blood, while they provoke irritated 
reactions from the hosts. When biting flies are present in high numbers it may affect health as 
well  as  productivity.  Sucking  flies  feed  on  sweat,  tears,  saliva,  urine,  faeces,  exudates  and 
blood from wounds. Sucking flies are in constant movement between all sorts of exudates and 
thereby  may  transmit  a  variety  of  diseases.  They  cannot  penetrate  the  skin,  with  their 
proboscis,  but  many  species  have  enlarged  prestomal  teeth  in  their  oral  aperture  which  can 
enlarge or even reopen nearly healed wounds.  

PT19 biocides against flies on grazing cattle, horses and other livestock can only claim to repel 
or attract (in order to trap) the target organism, not to prevent the diseases.  

5.6.5.7.1.1 Biology 

Hydrotaea  spp.  are  sucking  flies,  often  to  be  seen  in  the  eye  region,  around  the  udder,  and 
around  both  new  and  nearly  healed  lesions  where  they  seek  all  sorts  of  exudates.  Hydrotaea 
cannot  penetrate  the  skin  but  have  prestomal  teeth  on  labellum  which  can  enlarge  wounds. 
Hydrotaea irritans (head fly) is an important fly from an animal health point of view. It deposits 
its eggs in the ground in hedges and scrubs with high grass. The larvae are carnivorous, eating 
other  insect  larvae  in  the  ground.  It  normally  has  one  generation  per  year.  The  larvae 
overwinter  and  pupate  in  late  winter  and  hatch  into  adult  flies  in  late  June.  Other  important 
species  of  sucking  flies  are  Morellia  spp.,  Musca  domestica,  Musca  autumnalis,  which  develop 
directly  in  cattle-manure  and  have  several  generations  per  year.  These  sucking  flies  must  be 
seriously  considered  when  repellence  is  claimed  as  they  all  have  the  potential  to  transmit 
mechanically  the  diseases,  e.g.  M.  autumnalis  transmit  parafilaria,  and  Hy.  irritans  transmit 
summer mastitis.  

Several  biting  flies  such  as  horn  flies  Haematobia  irritans  and  Haematobia  stimulans  are  very 
common on cattle. These species develop in cattle manure, which means that these biting flies 
are  normally  only  seen  on  horses  when  the  horses  are  grazing  close  to  pastured  cattle.  They 
are both obligate blood-feeders, and primarily attack cattle and horses for a blood meal. They 
both have several generations during the summer. The horse fly Hypobosca equina is found in 
some  areas  mainly  on  horses,  but  also  from  time  to  time  on  cattle.  The  stable  fly  Stomoxys 
calcitrans also belong to the group of biting flies but breed in fermenting organic matter mixed 
with different kinds of manure and is, therefore, more common close to barns and cowsheds. A 
specific chapter is concerned with the stable fly indoor. 

Tabanids (horse flies, deer flies and clegs) are undoubtedly a very important reason for using 
repellent on horses, and cattle can also be heavily affected by the painful bite of tabanids. The 
small blood leaking lesions they make are attracting sucking flies and these can even be seen 
trying to push the horseflies aside to get access to the leaking blood themselves. Big tabanids 

 
 
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may only be seen in huge numbers for a short while and are therefore difficult to use as test 
species.  The  horse  fly  Haematopota  pluvialis  on  the  other  hand  is  often  very  abundant 
throughout the summer in permanent and old grazing areas, which has neither been ploughed 
nor fertilised. The bite of Tabanids is painful and causes considerable disturbance to horses and 
cattle  as  well  as  to  people  working  horses.  All  the  above-mentioned  flies  live  in  close  contact 
with their host and frequently land on them, which makes them vulnerable for treatment with 
repellents. 

Apart from these  species, there is  a wide  range of botflies which attack livestock. Botflies are 
obligate  parasites  and  therefore  always  need  a  host  for  larval  development  and  may  cause 
serious  damage  as  endoparasites.  These  are  flies  like  horse  bots  (Gasterophilus  spp.),  sheep 
bot  fly  (Oestrus  ovis)  or  warble  fly  (Hypoderma  spp.)  which  causes  considerable  disturbance 
among grazing animals, when they are present. The larvae of horse bots are endoparasites in 
horses  while  the  ox  warble  fly  is  an  endoparasite  in  cattle.  The  adult  female  flies  of  these 
botflies never get in contact with the horse coat as they spray eggs on the host’s coat without 
landing. On horses, eggs are often found attached to the forelegs but can also be seen on the 
mane  or  even  the  flanks.  Horses  take  the  fly  larva  in  when  licking  and  the  larva  develops  in 
tongue  epithelium  and  later  the  stomach.  The  ox  warble  fly  deposits  its  eggs  on  cattle 
(preferably  on  legs)  and  from  there  the  larvae  penetrate  through  the  skin,  develop  in  the 
connective  tissue,  and  end  up  causing  swellings  called  "warbles”  on  the  skin  surface.  As  the 
botflies do not land, it means that any repellence is difficult to measure  and methods must be 
developed in each case. 

One last important group of flies in this context is blowflies (Calliphoridae). The female of some 
of  these  species  like  Lucilia  spp.  seek  carcasses  for  feeding  and  egg  laying  and  the  larvae 
develop in the carrion. Sometimes though, blowflies lay eggs in living tissue in cattle or horses 
and the larvae may develop in wounds or in body cavities. As these flies land on the hosts they 
may be liable to a repellent. 

5.6.5.7.2 Dossier requirements  

Dossier requirements are stated in the Introduction (see section 5.6.5.1.3). 

5.6.5.7.2.1 Test species  

Few  of  these  fly  species  can  be  kept  in  captivity,  and  probably  none  can  act  biologically 
relevant  in  captivity  when  sensitive  biological  parameters  such  as  host-seeking  are 
investigated.  The  important  issue,  when  the  efficacy  of  a  repellent  is  measured,  is  whether  a 
product,  when  applied  to  the  host  in  a  relevant  environment,  can  overrule  all  the  key  stimuli 
that attract flies to their host. Tests with laboratory colonies of relevant species as well as with 
field collected specimens are applicable, provided such testing is justified in detail. 

Products  intended  for  use  as  a  repellent  on  cattle,  horses  and  small  ruminants  or  spatial 
repellents should be tested using the claimed host. A general host claim is not acceptable. 

Products  intended  for  use  as  a  repellent  against  specifically  claimed  fly  species,  e.g.  a 
Gasterophilus species must be tested with the species claimed, and efficacy against this species 
must be demonstrated.  

Spatial  or  topical  repellent  products  intended  as  a  general  fly-repellent  claimed  to  protect 
cattle,  horses  or  other  livestock  against  groups  of  nuisance  flies  (sucking  flies,  biting  flies, 
tabanids,  botflies,  blowflies)  must  be  tested  with  representative  species  for  the  claimed  host. 
The relevance of target fly species per host animal should be appropriately justified. For cattle 
and/or horses, the following species may be suggested as they are relatively easy to identify, 
but local adjustments using other fly species are acceptable if well argued: Sucking flies of the 
genus  Hydrotaea,  Musca  and  Morellia,  the  biting  flies  Haematobia  irritans  (which  can  be 
omitted  on  horses,  if  the  horses  are  grazing  isolated  from  cattle),  and  the  horse  flies 
Haematopota pluvialis. In some areas, other tabanids are more relevant and it is acceptable to 
use them instead if it is well argued in the report. 

A  general  claim  for  use  on  the  claimed  host  animal  can  only  be  given  if  efficacy  against  all 
groups is proven. 

In case  of specific claims,  e.g.  effective at high temperature, in  contact with  water, when the 
hosts  are  sweating,  when  horses  are  at  work,  etc.,  efficacy  must  be  demonstrated  in  the 
relevant situations. 

 
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It  must  be  clearly  described  in  the  report  how  the  test  method  ensured  that  the  claimed  fly 
species were counted in the field experiment. 

5.6.5.7.2.2 Requirements per type of claim and test methods 

A laboratory test is not required for product authorisation but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided. 

5.6.5.7.2.2.1 Products intended for use as repellents 

The efficacy of a product should be shown in either a robust simulated-use test, where animals 
can be tethered and observed close by or a field test where the animals are loose/untethered 
and  observed  from  a  distance  to  establish  the  efficacy  and  persistence  of  the  product  when 
used according to the label in a real-life situation.  

For PT19 products which are applied on animals, no common protocols are available. However, 
the EMA has published several guidelines for the evaluation of ectoparasiticides, see Appendix 
18.  These  guidelines  may  be  adapted  for  the  demonstration  of  the  efficacy  of  repellent 
products to be applied on animals, but any well documented protocol can be used, provided it 
is scientifically sound.  

It is important to give a detailed description of the circumstances in which the experiments are 
conducted, with special emphasis on the ratio of treated to untreated host animals at each trial 
location and trial replicate. It may be of consequence for the trial outcome whether all hosts in 
a herd are treated, compared to a situation where only a minor part of the hosts in a herd are 
treated. It is also of importance that the daily variation of fly activity is taken into consideration 
in  the  setup  of  the  efficacy  trials  and  reported  in  detail.  If  the  product  is  part  of  a  push-pull 
strategy  or  a  spatial  repellent,  e.g.  hosts  with  repellent  combined  with  attracting  traps,  then 
such a setup must be reported in detail as well. 

If treated horse rugs or similar covers are claimed to be effective in the field, then it must be 
demonstrated effective in the field. It is not important to make an evaluation of the flies on the 
rug itself, but the emphasis must be on the exposed parts of the host such as the neck, head, 
legs  and  belly,  including  the  sheath  on  males  and  udder  on  females,  which  are  thin-skinned 
areas and very attractive to several blood-feeding species. 

Tests must be presented with all host animal species claimed. Bridging efficacy  data from one 
host  species  to  another  can  be  accepted  based  on  robust  scientific  justification  taking  into 
account e.g. the difference in dosing between species (a small goat vs. a large cow), whether 
sweating is possible or not.  

5.6.5.7.2.2.1.1 Laboratory test 

Laboratory tests of repellents are not advised, as the differences in food seeking biology of the 
relevant species is huge.  

5.6.5.7.2.2.1.2 Simulated-use test  

In a simulated-use test different parameters are controlled. A simulated-use test of a repellent 
with full control of host animals and absence of their normal avoidance behaviour can be used 
to evaluate the efficacy and the persistency of the treatment; topically, spatial or impregnated 
textiles. The product should be applied according to the claim. Environmental conditions must 
be  specified  at  the  beginning  and  during  the  test  (temperature,  humidity,  photoperiod).  No 
specific temperature or humidity are recommended, but it must be warm and not too windy to 
have  fly  activity  high  enough  to  challenge  the  product.  A  control  treatment  without  repellent 
should be included in all trials to secure a measure of fly activity.  

It would be possible to  do simulated-use tests  e.g. an animal-to-cage  method similar to arm-
in-cage, with stable flies (S. calcitrans), house flies (M. domestica) and horn flies (Ha. irritans) 
as these can be kept in laboratories. Therefore, specific claims on these species may be backed 
by data generated in simulated use experiments, but it will not be possible to extrapolate such 
data to cover any other species encountered on grazing livestock. 

A simulated-use test must be conducted at a time in the season, as well as on days where the 
relevant  fly  species  are  active  in  the  field.  Depending  on  the  target  animal  species,  several 

 
 
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factors,  e.g.  hair  length,  thickness  of  the  coat,  grooming,  etc.  might  impact  the  efficacy  of 
topical  repellents.  Explain  in  the  test  report/documentation  the  circumstances  around  the 
choosing  of  the  test  animals.  If  animals  of  different  colour  are  chosen  be  sure  not  to  have  a 
majority of light-coloured animals. Ideally light coloured animals are not included, but state on 
the  label  if  the  product  is  for  a  specific  colour  of  animals,  if  only  light  coloured  animals  are 
tested a sound justification is needed to include all fur colours in the claim. For products only 
tested  on  e.g.  pied  (black  or  red)  cattle  it  can  be  necessary  to  state  on  the  label  that  the 
product  is  for  pied  (black  or  red)  cattle.  For  products  tested  on  sheep  it  can  be  necessary  to 
test on both  sheared and unsheared sheep (a sound justification is needed if  only sheared  or 
unsheared sheep are tested). 

At least two groups of host animals must be used, one group treated with the product and one 
group  with  no  treatment.  At  least  ten  different  individuals  are  suggested  to  be  used  in  each 
group. In the case of horses, a setup could be 2x5 horses with a treatment switch after e.g. a 
week (depended  on the residual efficacy  of the tested product) tested at one location,  or 2x5 
animals  tested  at  two  different  locations.  At  the  end  of  the  test  period,  there  must  be  2x10 
valid datasets. Each treatment group should be housed in separate enclosures throughout the 
course of the trials to prevent cross infestation or cross-contamination.  

Preferably, no test animals should have a recent history of treatment with insecticide/acaricide 
or  antiparasitic  agents.  Concerning  endo-antiparasitic  agents  this  is  acceptable,  but  details 
(product,  dose,  application  rate,  application  method,  date  of  the  last  treatment,  and  residual 
efficacy) must then be given in the report; the controls and the treated groups must have the 
same status. A safety margin for externally applied anthelmintics is the end of last application 
+1 month. The history of the treated and the control groups should be comparable. 

Livestock should be treated with the test product once the infestation has become established. 
The infestation should be documented by e.g. recordings of the numbers of flies, photographs. 
Dependent  on  the  circumstances  tests  can  be  conducted  using  treated  groups  in  comparison 
with untreated groups. If the product is acting at very close range it may be possible to make 
the  evaluation  by  treating  parts  of  the  host,  like  one  treated  side  of  the  neck  compared  with 
the equivalent area on the other, untreated, side of the neck. The advantage of such a setup is 
the neutralisation of individual host differences in fly-attraction. For dose determination, it may 
be  preferable  to  use  tethered  hosts.  The  attractiveness  of  the  test  animals  or  experimental 
groups should be tested prior to treatment. 

Percent  inhibition  (landing  or  feeding)  is  based  on  the  number  of  flies  landing/feeding  on  the 
untreated  control  animals  (or  room)  and  the  number  of  flies  landing/feeding  on  the  treated 
animals (room): 

Pct inhibition: ((LandingFeedingControl–LandingFeedingTreated)/LandingFeedingControl)x100. 

Alternatively  or  in  addition  to  fly  landing  catches,  also  the  frequency  of  certain  horse’s 
avoidance  behaviour  according  to  Mottet  et  al.,  see  Appendix  18,  can  be  recorded  as  a 
substitute, provided the abundance of the target species predominantly causing the avoidance 
behaviour  is  measured  at  the  beginning  and  the  end  of  the  test.  Test  designs  may  also  be 
adapted from Japin, M. and Haanen, G. A. Y., see Appendix 18. 

5.6.5.7.2.2.1.3 Field trials 

In  a  field  trial,  the  animals  must  have  the  opportunity  to  display  normal  avoidance  behaviour 
considering how the product is intended to be used. The tests may be conducted as described 
for  the  simulated-use  test  above,  with  the  adjustment  that  there  is  no  interference  with  the 
host animal’s normal behaviour in the field.  

The field trials should be conducted on two different geographical locations. 

At least two groups of host animals must be used, one group treated with the product and one 
group  with  no  treatment.  At  least  ten  different  individuals  are  suggested  to  be  used  in  each 
group.  Due  to  feasibility  and  cost-effectiveness,  it  would  be  possible  to  use  10  animals  one 
week (5 each in the treatment and control groups) and then use the same individuals again in 
the  second  week,  but  swap  treatments.  Each  animal  would  then  act  as  its  own  control. 
However,  any  residual  activity  of  the  product  has  to  be  excluded.  The  field  trials  can  be 
performed twice with 5 animals (in each of the control and treatment groups) each time at the 
same location, or with 5 animals (in each of the control and treatment groups) at two different 
locations.  At  least  10  animals  (each  as  control  and  treatment)  must  be  tested.  The  two 

 
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locations can be in the same country or region, e.g. two villages. In the case of cattle, the test 
can  be  performed  at  only  one  location  using  10  cows  for  the  control  and  10  cows  for  the 
treatment 

As  documentation  for  persistency,  counts  are  repeated  with  chosen  intervals  of  lengths 
dependent on the claim. If there is a claim for use on wet/sweating hosts, then data must be 
provided  showing  that  the  product  is  effective  under  such  circumstances.  The  guideline  from 
the  EMA:  “Guideline  on  specific  efficacy  requirements  for  ectoparasiticides  in  cattle”,  and 
Herholz et al., see Appendix 18, may be of inspiration for field trial setup. 

A field experiment to identify the level of efficacy of a product intended to protect cattle, horses 
or other livestock against flies must cover the following issues: 

Locality: Preferably low-lying areas with shelter. Important to establish double fencing between 
the  herds  to  prevent  contact  between  treated  and  untreated  hosts.  Preferably  treated  and 
control herds should be interchanged during a trial period to minimise differences between the 
two paddocks/fields. 

Herds:  Two  individual  experiments  each  constituting  a  treated  and  an  untreated  group.  The 
choice  of herds is dictated by the need of herds with neighbouring herds suitable as controls. 
Each group must constitute at least ten individuals. For horses a minimum of five individuals is 
acceptable.  

Census:  The  fly  activity  may  be  measured  through  the  summer,  preferably  on  windless,  hot 
days with high humidity. Prior to launching experiments monitoring of fly infestation should be 
performed. Observations of flies must be made for the time period relevant for the relevant fly 
species.  The  insect  activity  on  the  two  groups  of  hosts  (treated  and  untreated)  must  be 
counted within the frame of one hour, where environmental conditions are comparable for both 
groups. Each time the number of flies must be counted on the herd. Observations are made in 
five  different  areas  on  one  side  of  the  host’s  body:  at  the  head,  back,  side,  belly,  and  legs. 
Within  each  region,  the  number  of  flies  belonging  to  one  of  the  test  species  is  recorded.  The 
chosen test fly species are counted by direct observation. The observer must walk around the 
herd  to  minimize  the  influence  on  the  recordings  of  heterogeneous  fly  distribution  caused  by 
sunlight or breeze. The frequency of measures is dependent on the claimed persistency of the 
product.  

Insects:  The  following  species  may  be  suggested  as  they  are  relatively  easy  to  identify,  but 
local  adjustments  are  acceptable  if  well  argued:  Sucking  flies  of  the  genus  Hydrotaea,  and  of 
the genus Morellia, the biting flies Haematobia irritans (which can be omitted on horses, if the 
horses  are  grazing  isolated  from  cattle),  and  the  horse  flies  Haematopota  pluvialis.  In  some 
areas,  other  Tabanids  are  more  relevant  and  it  is  acceptable  to  use  them  instead  if  it  is  well 
argued in the report. 

The presence of botflies must be recorded when observed, but they are not very abundant, and 
the  figures  cannot  be  used  in  statistics.  If  a  claim  on  botflies  is  sought  it  may  be  possible  to 
evaluate  their  activity  by  the  number  of  eggs  on  the  skin,  but  a  method  is  not  described  for 
this, and therefore, if done, it must be explained in explicit detail. 

For  a  general  claim  against  groups  of  flies  (sucking  flies,  biting  flies,  Tabanids,  botflies  or 
blowflies), the species relevant for the claimed group (on the claimed host) must be present in 
sufficient  numbers  to  be  able  to  make  a  meaningful  and  statistically  significant  distinction 
between  treated  and  control  groups.  If  efficacy  has  been  shown  against  all  groups  a  general 
claim  for  use  on  the  claimed  host  animal  can  be  made.  For  a  specific  claim,  all  (individually) 
claimed fly species must be present in sufficient numbers. 

5.6.5.7.2.2.2 Attractants without PT18 active substances 

Use of an attractant in  a trap in  a push-pull setup may be suggested. A trap could be loaded 
with CO2, heat, octenol, butyric acid, black colour ball, etc. In such a setup the fly load will be 
measured as described above. 

For  product  authorisation  purposes,  the  efficacy  of  an  attractant  in  a  trap  without  an  active 
substance according to PT18 should be proven in a simulated use test or a field test according 
to the instructions for use. 

 
 
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The  attractants,  e.g.  the  traps,  used  for  the  test  should  be  identical  to  the  product  to  be 
marketed (see Introduction, section 5.6.5.1.3.7). Traps  should be tested on their  own,  with a 
control tested separately in a similar test setting.  

5.6.5.7.2.2.3 Attractants in combination with PT18 bait products 

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods). 

5.6.5.7.3 Assessment of authorisation 

5.6.5.7.3.1 Norms and criteria 

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for flies on grazing cattle, horses and other livestock in 
the following way: 

Products intended for use as repellent on animals (including a delivery system) 

A  simulated  use  test  or  a  field  test  demonstrating  ≥80%  repellency  within  the  test  period 
(according  to  the  claim),  directly  after  product  application  and  at  the  end  of  the  claimed  use 
period. 

All  claimed  target  host  animals  must  be  tested.  Claims  on  herds  as  well  as  individuals  are 
acceptable and should be tested as such. 

In case  of specific claims,  e.g.  effective at high temperature, in  contact with water, when the 
hosts are sweating, when horses are at work, etc. these should be demonstrated. 

Products  intended  for  use  as  a  spatial  repellent  or  for  use  as  attractants  without  PT18  active 
substances 

A  trap  with  an  added  attractant  should  catch  significantly  more  flies  than  one  without  the 
attractant. The traps used for the test should be identical to the product to be marketed.  

A  simulated  use  test  or  a  field  test  demonstrating  ≥  80%  efficacy  within  the  test  period 
according  to  the  claim,  directly  after  product  application  and  at  the  end  of  the  claimed  use 
period.  

For  attractants  at  least  a  ratio  of  4:1  of  test  individuals  trapped  in  the  trap  with  attractant 
compared  to  the  control  trap  within  the  test  period  (or  according  to  the  claim),  from  the 
beginning and until the end of the claimed efficacy period. 

All claimed target species must be tested. 

The  product  label  and  SPC  should  state  that  the  entire  insect  population  might  not  be 
caught/repelled  by  this  type  of  product;  the  label  claim  should  be  “reduces”  and  not 
“protects”/”protection”, with no mention of a CPT. 

5.6.5.8 Fruit Flies and Scuttle Flies 

5.6.5.8.1 Introduction  

The purpose of biocidal repellents or attractants for fruit flies and scuttle flies is to prevent and 
fight an infestation of food, domestic waste or medical and veterinary facilities. 

Fruit flies may cause inconvenience primarily indoors where they infest rotting organic material 
such as fermenting fruits and vegetables.  

In Europe, species of the family Drosophilidae (fruit flies or vinegar flies) can be considered as 
a relevant nuisance. 

Fruit fly: Drosophila spp., e.g. Drosophila melanogaster. 

Within  the  family  Phoridae  (scuttle  flies,  humpbacked  flies  or  coffin  flies),  the  introduced 
species  Megaselia  scalaris  may  cause  inconvenience  or  hygienic  problems  primarily  indoors 
while infesting food or wounds. 

Scuttle fly: Phoridae, e.g. Megaselia scalaris 

 
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5.6.5.8.1.1 Biology  

The life cycle of both fruit flies and scuttle flies contains four developmental stages: egg, larva 
(maggot), pupa and imago (adult). Most species of fruit flies have a saprophagous lifestyle, as 
both  larvae  and  adults  are  feeding  on  decaying  matter.  Eggs  are  laid  on  rotting  organic 
material  including  fermenting  fruits  and  vegetables.  Once  hatched,  larvae  feed  by  burrowing 
into the organic debris and filtering decaying organic matter. They then undergo transformation 
into the imago via a pupal stage. Adults feed by regurgitating on food and then taking up the 
pre-digested  food  in  liquid  form.  The  life  cycle  of  fruit  flies,  from  egg  to  fly,  is  1  to  3  weeks, 
depending on the climate conditions (shortest: 7 days at 29°C). The adult fruit flies have a life 
expectancy of about 40 to 50 days.  

Fruit flies regularly fly into and  out  of man-made  structures. Inside,  fruit flies land  on human 
food  or  organic  waste.  Fruit  flies  play  only  a  minor  role  in  spreading  pathogens  since  they 
normally  are  not  in  contact  with  decaying  or  infective  animal  products,  e.g.  flesh,  carcasses, 
faeces.  They,  therefore,  do  not  pose  a  serious  health  risk;  however,  severe  infestations  can 
render  the  food  unfit  for  human  consumption,  due  to  a  common  averseness  against  infested 
food and due to acceleration of fruit decay.  

However, scuttle flies are ecologically diverse and include saprophagous as well as parasitic or 
omnivorous  species.  The  larva  of  M.  scalaris  feeds  on  a  broad  variety  of  decaying  organic 
material and adults may also land on open wounds of patients in medical facilities  in order to 
lay eggs (wound myiasis). Potentially, pathogens can be transferred to wounds from faecal or 
other decaying material. The development time of the Megaselia species is similar to that of D. 
melanogaster. 

The differentiation of fruit flies and scuttle flies may be problematic for the general public since 
they are similar in size and habitus. 

5.6.5.8.2 Dossier requirements  

Dossier requirements are stated in the Introduction (see section 5.6.5.1.3). 

5.6.5.8.2.1 Test species  

A  product  claimed  “against  fruit  flies”  has  to  be  tested  on  D.  melanogaster  and  a  product 
claimed  “against  scuttle  flies”  has  to  be  tested  on  M.  scalaris.  Adult  organisms  should  be 
tested,  deviations  should  be  justified  and  tested,  i.e.  if  other  developmental  stages  are 
claimed. For a general claim against “fruit and scuttle flies” both species, D. melanogaster and 
M. scalaris have to be tested. For a species-specific claim, testing against the claimed species is 
required. 

Due to the specificity of certain active substances, e.g. pheromones, for products based on an 
active substance with a species-specific mode of action, only effects against fruit fly or scuttle 
fly  species  that  have  been  tested  under  simulated-use  and/or  field  conditions,  depending  on 
the type of claim, should be claimed on the product label and in the SPC. 

5.6.5.8.2.2 Requirements per type of claim and test methods 

A laboratory test is not required for product authorisation but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided. 

5.6.5.8.2.2.1 Repellent products as space and/or surface treatment 

For product authorisation purposes the efficacy of repellent products should be proven in: 

•  a  simulated-use  test  according  to  the  instructions  for  use  (test  design  example  see 

5.6.5.8.2.2.1.2), or  

•  a  field  trial  according  to  the  instructions  for  use  (test  design  example  see 

5.6.5.8.2.2.1.3). 

Products applied onto surfaces may act either by evaporation or on the surface itself. 

For products applied on surfaces, two porous and one non-porous surface should be used, e.g. 
ceramic  tile,  plywood,  painted  plywood,  stainless  steel,  concrete,  for  a  general  label  claim  as 

 
 
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“surface treatment”. The efficacy of each surface should be proven in a separate test, i.e. three 
tests for three different surfaces. For authorisation of a product to be used on a specific type of 
surface the efficacy for only this specific surface should be assessed. 

The repellents used for the test should be identical to the product to be marketed. 

If  outdoor  use  is  claimed,  the  treated  surface/substrate  or  repellent  product  and  control 
surfaces/substrates  have  to  be  kept  outside  to  ensure  adequate  weathering  and  the  outdoor 
conditions must be specified (for details see Introduction, section 5.6.5.1.4.1.3). 

For products intended to be used as  space treatment the room size stated in the SPC  and on 
the  label  must  be  used  in  the  efficacy  trial.  For  the  extrapolation  to  larger  room  sizes  than 
proven in the efficacy test a justification is necessary. 

5.6.5.8.2.2.1.1 Laboratory choice test 

Different set-ups are recommended for testing the repellency of adult flies and larvae. Only one 
appropriate set-up is required per use. 

For each of those tests the following requirements have to be fulfilled: 

•  at least 5 replications and 5 negative controls should be used; 

•  environmental conditions must be specified for the test itself and during the storage of 
the treated surfaces/substrates or repellent product (temperature, humidity, ventilation, 
and photoperiod); 

• 

for residual efficacy, flies shall be exposed to the product at several time intervals after 
application (including the end of the claimed period). 

5.6.5.8.2.2.1.1.1 Trap Bioassay (see Appendix 18: Knaden M., et al.) 

Transparent  test  chambers  (approximately  10x8x10  cm)  are  equipped  with  a  treatment  trap 
and a control trap, each in one corner. The trap position should rotate to avoid site preference. 
The  traps  could  be  made  from  small  cups  or  containers  (30  ml)  with  a  lid.  The  lids  contain  a 
hole to insert a cut micropipette tip (tip diameter 2–3 mm), that the narrower part is pointed 
toward  inside.  An  attractive  food  source,  preferably  Drosophila  fly  food  (see  Appendix  18: 
Alcaine-Colet,  A.,  et  al.),  is  applied  in  both  traps.  Whereas  the  repellent  is  only  added  in  one 
trap. 50 flies (sex ratio 1:1; 4–5 days old; not older than 7 days as aging affects the sensory 
abilities)  that  are  starved  for  24  hours  before  the  experiments  with  water  ad  libitum,  are 
transferred  into  the  test  chambers.  The  number  of  flies  in  and  outside  the  traps  should  be 
counted. 

5.6.5.8.2.2.1.1.2 Choice  assay  using  the  “capillary  feeder”  (CAFE)  method  (see 
Appendix 18: Ja, W.W., et al.) 

The set-up consists of two transparent chambers, an inner chamber containing the flies and an 
outer  chamber,  filled  with  water.  The  inner  chamber  could  be  prepared  by  paring  down  a  1.5 
cm diameter plastic vial to 2 cm length, with the bottom pierced to allow entry of water vapour 
and  air  from  the  outer  chamber.  Two  glass  micropipettes  filled  with  a  liquid  medium  by 
capillary  action  are  inserted  through  the  cap  via  truncated  pipette  tips.  Attractive  liquid  food 
could be topped with an oil layer to minimize  evaporation.  The repellent is  only added in one 
micropipette.  To  facilitate  visualization,  a  red  dye  could  be  added  to  the  medium  and  can  be 
seen  in  the  proboscis  and  abdomen  of  the  fly.  The  number  of  flies  feeding  on  both  pipettes 
should  be  counted.  The  set-up  can  be  conducted  with  flies  housed  individually  up  to  groups 
with eight animals per chamber. 

5.6.5.8.2.2.1.1.3 Double cage or glass tubes test 

Experiments  should  be  conducted  in  a  test  apparatus  consisting  of  two  cages  (approximately 
30x30x30  cm)  connected  by  a  tunnel.  Walls  should  be  transparent  to  allow  better  insect 
counting, and all surfaces should be of cleanable material, sealed and waterproof that is 100% 
decontaminated.  Both  ends  of  the  tunnel  are  equipped  with  a  gate  that  connects  the  tunnel 
with the cage. One cage contains the repellent whereas the opposite cage remains untreated. 
The application quantity of the repellent must be matched to the size of the boxes. According 
to  the  claim  the  floor  of  the  cages  should  be  of  e.g.  porous  and  non-porous  substrate 
representing  surfaces  typically  treated,  e.g.  ceramic  tile,  plywood,  painted  plywood,  stainless 
steel,  concrete. At the  start  of the  experiment, the repellent is applied, and 50 adult flies are 

 
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released in the middle of the tunnel with both gates closed. Alternatively, a third cage could be 
used  as  a  release  box  interconnected  by  two  tunnels  between  both  cages.  After  10  minutes, 
the gates are opened, and the insects can choose the treated or control cage. The number of 
flies in each test cage is recorded 5, 10, 30 and 60 minutes after exposure to the repellent. The 
ratio  of  insects  found  in  the  treated  vs.  control  cage  is  a  measure  for  the  efficacy  of  the 
product. Depending on the claim the test could be performed with or without food attractant. 

Alternatively, the cages could be replaced by glass tubes, usually, three tubes (approximately 
50  cm  length  each  and  a  diameter  of  at  least  10  cm);  insects  in  the  middle  tube  and  two 
peripheral  tubes,  one  containing  the  repellent;  peripheral  tubes  should  be  closed  by  gauze  to 
allow air circulation. 

The  test  apparatus  should  be  installed  in  a  ventilated  room  to  avoid  contamination  of  the 
control cage/tube by evaporation of the repellent. 

To  exclude  side  preference  effects  a  control  treatment  without  active  substance  in  any  cage 
should be conducted with insects from the same insect population.  

5.6.5.8.2.2.1.1.4 Choice bioassay for larvae (see Appendix 18: Dweck, Hany K.M., et al.) 

Petri dishes (diameter: approximately 10 cm), filled  with 1% agarose solution can be used as 
behavioural  arena.  The  dish  should  be  divided  into  two  equal  parts  and  two  filter  paper  discs 
(diameter:  0.5  cm)  are  applied  on  each  side  of  the  dish  at  the  periphery;  one  containing  the 
repellent. Transfer 50 larvae (2nd or 3rd instar) in the centre of the Petri dish. Number of larvae 
on each side should be counted. 

5.6.5.8.2.2.1.1.5 Choice  bioassay  for  testing  flies  oviposition  behaviour  (see  Appendix 
18: Dweck, Hany K.M., et al. and Stensmyr, Marcus C., et al.) 

Transparent test chambers (of approximately 50x50x50 cm) are equipped with two Petri dishes 
(diameter:  approximately  10  cm),  filled  with  oviposition  medium,  each  in  one  corner.  The 
repellent is only added in one Petri dish, 20 mated female flies (4–5 days old; not older than 7 
days as aging affects the sensory abilities) are transferred into the test chamber. The number 
of eggs in each Petri dish should be counted. 

5.6.5.8.2.2.1.2 Simulated-use test 

Mandatory requirements: 

•  A choice test should be conducted with an attractive food source. 

•  For ambient repellents for protection of large rooms, e.g. diffusers to protect rooms with 
a defined volume, a double room test should be performed, with at least one 20 m³ test 
room for product application. Such spatial repellent products can repel fruit and scuttle 
flies  to  protect  rooms  from  entering  and/or  dispel  the  insects  from  already  infested 
areas.  

•  Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative 

control) should be performed.  

•  Environmental conditions must be specified for the test itself  and during the storage of 
the 
repellent  product  (temperature,  humidity, 
photoperiod,  ventilation).  The  temperature  would  be  expected  to  fall  in  the  range  of 
20-28°C. 

treated  substrates/surfaces  or 

•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8). 

For  products,  having  a  limited  releasing  rate  into  the  air,  e.g.  the  following  set-up  could  be 
used.  Other  test  designs  than  the  following  examples  can  be  accepted  if  the  protocol  is 
scientifically valid. 

Test design: Test rooms are equipped with an attractive food source, preferably Drosophila fly 
food  (see  Appendix  18:  Alcaine-Colet,  A.,  et  al.),  presented  on  a  table  with  a  height  of 
approximately 0.5 m. Report the location of  the table in the  chamber. At least 200 adult free 
flying  flies,  D.  melanogaster  or  M.  scalaris,  mixed  sexes,  are  released  into  the  room.  After  at 
least 1 hour of acclimatization the repellent product is applied according to the  claim,  e.g.  on 
both porous and non-porous substrate representing surfaces typically treated e.g. ceramic tile, 
plywood, painted plywood, stainless steel, concrete. The number of flies should be monitored, 

 
 
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for  at  least  1  minute,  at  the  end  of  the  claimed  efficacy  period,  within  a  defined  radius,  e.g. 
minimum 40 cm radius on the surface where the food is presented  or according  to the claim, 
around  the  attractive  food  source.  For  residual  efficacy,  flies  are  exposed  to  the  product  at 
several time intervals after application (including the end of the claimed period). 

Proof  of non-insecticidal  efficacy: After  each test  run the mortality  of  the adult insects should 
be monitored. 

Efficacy assessment: The potential repellent effect of the product is determined by  comparing 
the number of flies at or around the attractive food source with the repellent (within a defined 
space) to the number of flies at or around the attractive control food source (negative control). 
Alternatively, the effect on the next generation can be used to determine the repellent efficacy. 
This can be conducted after the trial by maintaining the food source in a cabin/cage free of flies 
in order to evaluate if some larvae hatch after 2 to 4 days. The number of larvae and/or adults 
found on the treated vs. control food source (negative control) is a measure for the efficacy of 
the product (% offspring). 

5.6.5.8.2.2.1.3 Field test 

In the  field trials, the product is tested in the  actual use  situation, for instance in an infested 
home, medical and veterinary facilities or warehouse and applied according to the direction for 
use in the SPC. 

Test design: The test set-up should be similar to the one described for the simulated-use trial 
(see  5.6.5.8.2.2.1.2),  and,  for  vapour-based  products,  reflect  the  claimed  air  volume 
(according to the direction for use; at least 20 m³). 

Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative  control) 
should be performed.  

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  surfaces/substrates  or  repellent  product  (temperature,  humidity,  photoperiod, 
ventilation). The temperature would be expected to fall in the range 20-28°C. 

Proof  of  non-insecticidal  efficacy,  if  it  cannot  be  waived  (for  details  see  Introduction,  section 
5.6.5.1.3.8), has to be examined at the end of the trial. 

Efficacy assessment: The potential repellent effect of the product is determined by  comparing 
the number of flies at or around the attractive food source with the repellent (within a defined 
space) to the number of flies at or around the attractive control food source (negative control). 
Alternatively, the effect on the next generation can be used to determine the repellent efficacy. 
This can be conducted after the trial by maintaining the food source in a cabin/cage free of flies 
in order to evaluate if some larvae hatch after 2 to 4 days. The number of larvae and/or adults 
found on the treated vs. control food source (negative control) is a measure for the efficacy of 
the product (% offspring). 

5.6.5.8.2.2.2 Attractants without PT18 active substances  

For  product  authorisation  purposes  the  efficacy  of  attractants  in  traps  without  PT18  active 
substance should be proven in: 

•  a  simulated-use  test  according  to  the  instruction  for  use  (test  design  examples  see 

5.6.5.8.2.2.2.2), or  

•  a  field  trial  according  to  the  instruction  for  use  (test  design  examples  see 

5.6.5.8.2.2.2.3). 

The attractants, e.g. the traps, used for the test should be as similar as possible to the product 
to be marketed (for details see Introduction, section 5.6.5.1.3.7).  

If  outdoor  use  is  claimed,  the  test  should  be  performed  outdoors,  and  the  attractant  product 
and control have to be kept outside to ensure adequate weathering and the outdoor conditions 
must be specified (for details see Introduction, section 5.6.5.1.4.1.3.). 

5.6.5.8.2.2.2.1 Laboratory choice test 

Different  set-ups  are  recommended  for  testing  attractants  of  adult  flies  and  larvae.  A  choice 
test  similar  to  the  tests  described  in  section  5.6.5.8.2.2.1.1  could  be  used  by  comparing  the 

 
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number  of  flies  preferring  the  attractant  vs.  attractive  control  food  source,  preferably 
Drosophila fly food. 

5.6.5.8.2.2.2.2 Simulated-use test 

Mandatory requirements: 

•  The  simulated-use  test  should  be  conducted  in  a  room  of  at  least  20  m3  with  an 

attractive food source. 

•  Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative 

control) should be performed. 

•  Environmental conditions must be specified for the test itself and during storage of the 
attractant  product  (temperature,  humidity,  photoperiod,  ventilation).  The  temperature 
would  be  expected  to  fall  in  the  range  20-28°C.  Temperature  and  relative  humidity 
should be kept at a constant level (±1°C, ±10% relative humidity) throughout the test 
period and for all replicates. 

•  Traps  should  be  tested  on  their  own,  with  a  negative  control  tested  separately  in  an 

identical test setting. 

• 

If the product is claimed to protect premises without food sources, where flies originate 
from near infested areas, the efficacy should be tested without food source. 

Other test designs than the following examples can be accepted if the  protocol is scientifically 
valid. 

Test design: Test rooms are equipped with an attractive food source, preferably Drosophila fly 
food,  presented  on  a  table  with  a  height  of  approximately  0.5  m.  Report  the  location  of  the 
table in the chamber. At least 200 adult free flying flies, D. melanogaster or M. scalaris, mixed 
sexes,  are  released  into  the  room.  After  at  least  1  hour  of  acclimatization  the  attractant 
product/trap  is  applied  according  to  the  label  claim.  In  order  to  prove  the  choice  of  the  flies, 
the distance between food source and trap should be large enough to avoid accidental catching 
of  the  flies  due  to  undirected  movement.  As  the  distance  may  depend  on  the  product,  the 
distance used in the trial should be the same as claimed on the product label and in the SPC. 
Number  of  flies  is  monitored  at  defined  time  intervals  until  the  end  of  the  claimed  efficacy 
period,  e.g.  0.5  and  1  hour  after  introduction  of  the  product).  For  residual  efficacy,  flies  are 
exposed  to  the  product  at  several  time  intervals  after  application  (including  the  end  of  the 
claimed period). 

Efficacy assessment: Attraction may depends on the mode of action of pheromones. Therefore, 
attractants  may  be  sex  specific  or  sex  unspecific.  Attracting  effectiveness  of  a  sex  unspecific 
product is determined  by comparing the number  of flies within the treated trap to the control 
trap. For products which affect only individuals of one sex the efficacy should be shown on the 
next  generation.  This  can  be  conducted  after  the  trial  by  maintaining  the  food  source  in  a 
cabin/cage free of flies in order to evaluate if some larvae hatch after 2 to 4 days. The number 
of  larvae  and/or  adults  found  on  the  treated  vs.  control  food  source  is  a  measure  for  the 
efficacy of the product (% offspring). 

5.6.5.8.2.2.2.3 Field test 

In the  field trials, the product is tested in the  actual use  situation, for instance in an infested 
home, medical and veterinary facilities or warehouse and applied according to the direction for 
use in the SPC. 

Test design: The test set-up should be similar to the one described for the simulated-use trial 
(see  5.6.5.8.2.2.2.2),  and,  for  vapour-based  products,  reflect  the  claimed  air  volume 
(according to the direction for use; at least 20 m³). 

Any bycatch of non-target insects has to be recorded and identified at least to order, preferably 
to the family level. 

Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative  control) 
should be performed.  

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
attractant  product  (temperature,  humidity,  photoperiod,  ventilation).  The  temperature  would 
be expected to fall in the range 20-28°C. 

 
 
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Efficacy assessment: The potential attracting  effect of the product may depends on the mode 
of  action  of  pheromones.  Therefore,  attractants  may  be  sex  specific  or  sex  unspecific. 
Attracting effectiveness of a sex unspecific product is determined by comparing the number of 
flies within the treated trap to the control trap. For products which affect only individuals of one 
sex the efficacy should be shown on the next generation. This can be conducted after the trial 
by maintaining the food source in a cabin/cage free of flies in order to evaluate if some larvae 
hatch  after  2  to  4  days.  The  number  of  larvae  and/or  adults  found  on  the  treated  vs.  control 
food source is a measure for the efficacy of the product (% offspring). 

5.6.5.8.2.2.3 Attractants in PT18 bait products 

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for  bait 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods). 

5.6.5.8.3 Assessment of authorisation  

5.6.5.8.3.1 Norms and criteria  

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for fruit flies and scuttle flies in the following way: 

Products  intended  for  use  as  repellents  against  fruit  flies  and  scuttle  flies  as  space  and/or 
surface treatment: 

Non-insecticidal  efficacy  has  to  be  proven  in  a  simulated-use  test  if  it  cannot  be  waived  (for 
details see Introduction, section 5.6.5.1.3.8). 

The required results for the different trials are: 

Laboratory choice-tests (not required for product authorisation):  

•  ≥  80%  repellency  within  the  test  period  (according  to  the  claim),  from  the  beginning 

and until the end of the claimed efficacy period. 

Simulated-use test:  

•  ≥ 80% efficacy (depending on the assessment method (see 5.6.5.8.2.2.1.2), i.e. ≤ 20% 
offspring or ≥ 80% repellency) within the test period (according to the claim), from the 
beginning and until the end of the claimed efficacy period. 

Field test:  

•  ≥  80%  repellency  (depending  on  the  assessment  method  (see  5.6.5.8.2.2.1.3),  i.e.  ≤ 
20%  offspring  or  ≥  80%  repellency)  within  the  test  period  (according  to  the  claim), 
from the beginning and until the end of the claimed efficacy period. 

The product label and the SPC should state that the insect population may not be  reduced to 
zero  by  the  use  of  this  type  of  product  alone;  therefore,  the  product  should  be  used  in 
combination  with  other  products  or  technologies  as  part  of  integrated  pest  management.  The 
label claim should be “reduces population”, not “eliminates population”. 

Attractants without PT18 active substances  

The required results for the different trials are: 

•  at least a ratio of 4:1 of test individuals trapped in the trap with attractant compared to 
the control trap within the test period (or according to the claim), from the beginning 
and until the end of the claimed efficacy period.  

Laboratory choice-tests (not required for product authorisation):  

•  ≥  80%  attraction  compared  to  the  negative  control  within  the  test  period  according  to 

the claim, from the beginning and until the end of the claimed efficacy period. 

Simulated-use test:  

•  ≥ 80% efficacy (depending on the assessment method (see 5.6.5.8.2.2.2.2) i.e. ≤ 20% 
offspring  or  ≥  80%  trapped  test  individuals)  within  the  test  period  according  to  the 
claim (e.g. sex), from the beginning and until the end of the claimed efficacy period. 

 
 
 
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Field test:  

•  ≥ 80% efficacy (depending on the assessment method (see 5.6.5.8.2.2.2.3) i.e. ≤ 20% 
offspring  or  ≥  80%  trapped  test  individuals)  within  the  test  period  according  to  the 
claim, from the beginning and until the end of the claimed efficacy period. 

The  product  label  and  the  SPC  should  state  that  not  the  entire  insect  population  might  be 
caught by this type of product; therefore, the product should be used in combination with other 
products  or  technologies  as  part  of  integrated  pest  management.  The  label  claim  should  be 
“reduces population”, not “eliminates population”.  

5.6.5.9 Mosquitoes 

5.6.5.9.1 Introduction 

Mosquitoes, including different species of notably the genera Culex, Aedes, and Anopheles, are 
common pests in the EU. While mosquitoes are mainly a nuisance problem in most parts of the 
EU, some species are known for their vector competence, e.g. the Asian tiger mosquito (Aedes 
albopictus)  and  there  have  been  reports  of  local  dengue  transmission  in  France,  Croatia  and 
Spain. Chikungunya, another viral disease transmitted by mosquitoes, reached Europe in 2007 
resulting  in  a  local  outbreak  in  northern  Italy  with  almost  200  cases.  On  a  global  scale, 
mosquitoes are responsible for transmitting diseases such as malaria (Anopheles spp.), yellow 
fever  (Aedes  spp.,  mainly  Aedes  aegypti),  dengue  fever  (Ae.  aegypti  and  Ae.  albopictus), 
Chikungunya (Ae. aegypti and Ae. albopictus), Zika (Ae. aegypti, Ae. albopictus) and West Nile 
fever  (Culex  spp.).  So  far  most  of  these  exotic  pathogens  are  not  endemic  in  Europe,  but 
occasional  outbreaks  may  occur,  and  travellers  might  also  encounter  them  when  visiting 
disease-endemic  (tropical  or  subtropical)  countries  around  the  globe.  Nevertheless,  an 
increasing number of autochthonous infections has been reported for  West Nile virus, malaria 
and dengue since 2012. 

PT19 biocides against mosquitoes can only claim to repel or attract (in order to trap) the target 
organisms  and  to  protect  humans  and/or  animals  from  being  bitten,  but  not  to  prevent  the 
diseases. 

5.6.5.9.1.1 Biology  

Like  all  Diptera,  mosquitoes  go  through  four  development  stages.  The  egg,  larval  and  pupal 
stages take place in stagnant water bodies such as floodplains, drainage ditches, and natural or 
artificial water containers. Depending  on the genus,  female  mosquitoes lay their  eggs directly 
on the water surface, e.g. Culex spp., Anopheles spp., or above the water line, e.g. Aedes spp. 
on damp surfaces in areas that are likely to temporarily flood. Depending on the genera, eggs 
are laid individually, e.g. Aedes spp., Anopheles spp., or in bundles called rafts, e.g. Culex spp. 

Once  larvae  hatch,  filter  feeding  begins  near  the  water  surface.  Mosquitoes  go  through  four 
larval  instar  stages  before  entering  the  pupal  stage.  The  pupal  stage  lasts  about  two  days, 
during  this  time  mosquitoes  do  not  feed.  Once  completed,  the  adult  emerges  and  enters  the 
terrestrial environment. Mating begins a few hours to days after emergence. Once mated, the 
female starts to search for a blood meal. Humans, birds, and other animals are potential blood 
hosts, with some mosquito species preferring human blood to other animals. 

The  adult  female  mosquito  locates  her  host  through  visual  cues,  heat,  moisture,  and  most 
importantly  host-derived  odour  plumes  that  represent  a  strong  olfactory  cue  (exhaled  carbon 
dioxide and skin emanations). Once located, the host-seeking female mosquito will attempt to 
bite and eventually take up a blood meal. This blood meal is partially digested and used for the 
development of eggs. About three to four days after the blood meal, the female will lay 50 to 
500  eggs.  Most  species  lay  eggs  more  than  once  during  their  life  span,  thus,  shortly  after 
oviposition  females  will  start  to  seek  their  next  blood  meal.  This  particular  behaviour  turns 
some mosquito species into very effective vectors of disease. If they feed on an infected host, 
they  can  become  infectious  themselves,  provided  that  they  are  capable  vectors  of  the 
pathogen, and transmit the pathogen to their next host. 

5.6.5.9.2 Dossier requirements  

General dossier requirements are stated in the Introduction (see section 5.6.5.1.3). 

In order to eliminate the risk of disease transmission to human volunteers in field settings, field 
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on  humans  or  clothing  (for  details  see  Introduction,  section  5.6.5.1.3.4.3).  In  the  case  of 
products  applied  to  domestic  animals,  field  trials  can  be  less  stressful  for  the  animals  and 
should therefore be accepted instead of simulated-use test. 

For authorisation of products applied on human or animal skin efficacy must be proven with the 
recommended label dose and according to the  claimed mode  of application given on the label 
and in the SPC. The dose used in the efficacy studies should be covered by human health risk 
assessment. 

5.6.5.9.2.1 Test species  

Products intended for use as repellent for human or animal skin, clothing: 

For  a  general  claim  against  mosquitoes,  repellent  efficacy  testing  must  be  conducted  with  at 
least one mosquito species from each of the three genera Culex, Aedes and Anopheles. Culex 
spp. is the most common mosquito in Europe. Aedes spp. are the most aggressive mosquitoes.  

For a species-specific label claim, testing against the claimed species is required.  

When use against tropical mosquitoes or use in tropical areas is claimed, it should be specified 
against which mosquito spp. the  product is effective and these should be tested. The minimal 
efficacy should be proven against three genera: Aedes spp., Culex spp. and Anopheles spp. The 
choice of test species should be justified. 

Products intended for use as general surface and spatial repellents: 

For  a  general  claim  against  mosquitoes,  repellent  efficacy  testing  must  be  conducted  with  at 
least  one  mosquito  species  from  each  of  the  two  genera  Culex  and  Aedes.  Culex  spp.  is  the 
most common mosquito in Europe. Aedes spp. are the most aggressive mosquitoes.  

For a species-specific label claim, testing against the claimed species is required.  

When  use  against  tropical  mosquitoes  or  use  in  tropical  areas  is  claimed,  repellent  efficacy 
testing  must  be  conducted  with  at  least  one  mosquito  species  from  each  of  the  three  genera 
Culex, Aedes and Anopheles. 

General requirements for all products against mosquitoes: 

Due to the specificity of certain active substances, e.g. pheromones, for products based on an 
active  substance with a species-specific mode  of action,  only effects against mosquito  species 
that  have  been  tested  under  simulated-use  and/or  field  conditions,  depending  on  the  type  of 
claim, should be claimed on the product label and in the SPC.  

For insects from laboratory rearing used in the efficacy studies the test species, strain and age 
should  be  reported  (for  more  details  see  Introduction,  section  5.6.5.1.3).  All  test  mosquitoes 
should be non-blood fed, but not starved. Sugared water has to be provided before and during 
the test in order to keep the mosquitoes fit for testing. 

5.6.5.9.2.2 Requirements per type of claim and test methods 

A laboratory test is not required for product authorisation but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided. 

5.6.5.9.2.2.1 Products intended for use as topical repellents for human skin 

For  product  authorisation  purposes  the  efficacy  of  repellent  products  targeting  the  use  on 
human skin should be proven in a simulated-use test according to the instructions for use (test 
design example see 5.6.5.9.2.2.1.2).  

The repellents used for the test should be identical to the product to be marketed. 

CPT  values must be determined in a  simulated-use test. As field studies are not  evaluated as 
key studies, data from the field studies cannot be used to determine CPT values. 

5.6.5.9.2.2.1.1 Laboratory test 

Different set-ups are recommended for testing repellent products targeting the use on human 
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For each of those tests the following requirements have to be fulfilled:  

•  at least 10 replications and 10 negative controls should be used; 

•  environmental  conditions  must  be  specified  for  the  test  itself  (temperature,  humidity, 
ventilation,  and  photoperiod).  The  temperature  would  be  expected  to  fall  in  the  range 
between 19-29°C. 

5.6.5.9.2.2.1.1.1 Olfactometer test  

Y-tube  olfactometer  tests  can  be  performed  to  measure  the  behavioural  responses  of  host-
seeking  female  mosquitoes  towards  volatile  repellent  active  ingredients  in  the  presence  of 
natural host odours, see Appendix 18: Geier M. and Boeckh J.. Each olfactometer consists of a 
transparent  acrylic  glass  base  leg,  followed  by  a  decision  chamber  and  two  branches  which 
terminate into Teflon chambers, where the test stimuli are introduced. The system is supplied 
with a constant purified air stream heated up to 26±1°C and humidified to a relative humidity 
of 70±5%. Rotating doors in both branches, as well as at the downwind end  of the base leg, 
allow the release and entrapment of the test mosquitoes. Cohorts of 15-20 host seeking female 
mosquitoes are attached to the apparatus at its downwind end.  

Repellent test formulations are applied on filter paper strips (approximately 1 cm×3 cm) which 
can be suspended into one of the Teflon chambers. A forefinger or a hand is then inserted into 
the  Teflon  chamber  behind  the  paper  strip  and  the  rotating  door  of  the  base  leg  is  opened. 
Mosquitoes are allowed to fly upwind for 30 seconds and decide  between the test branch with 
volatile stimuli and the control branch with a finger or a hand without repellent. At the end of a 
test,  the  rotating  doors  are  closed  and  the  number  of  mosquitoes  that  migrated  from  the 
release cage (= active), the number of mosquitoes inside the test cage (where the stimuli were 
applied), and the number of mosquitoes in control cage (with filtered air) are documented. At 
the conclusion of a test, the airflow in the apparatus is inverted and mosquitoes are lured back 
into  the  release  cage  by  the  palm  of  the  hand  and  the  next  of  four  y-tubes  can  be  used  for 
testing. Treatments are tested in randomized order, and after each run, the control branch and 
test branch are changed to avoid position or adaptation effects. Each single compound or mix 
are tested in 10 replicates. All treatments are  tested against a control of the forefinger and a 
paper strip treated with solvent only. 

5.6.5.9.2.2.1.1.2 Blood Feeding Assays (see Appendix 18: Mulatier M., et al.) 

The set-up consists of a container (approximately 10 cm length and 7 cm diameter) containing 
25  female  mosquitoes  and  covered  with  a  piece  of  polyester  net  previously  impregnated  with 
the  repellent.  Containers  are  placed  on  a  membrane  allowing  the  mosquitoes  to  feed  through 
it. After 1 h of exposure, the proportion of blood-fed females can be counted. 

5.6.5.9.2.2.1.2 Simulated-use test 

In  this  test,  the  repellent  is  applied  to  the  skin  of  the  forearm  of  a  human  volunteer  and 
regularly  exposed  to  caged  populations  of  host-seeking  pathogen-free  mosquitoes.  Different 
set-ups can be used: “arm-in-cage”, “arm-to-cage” or a room test.  

In  all  set-ups  a  minimum  landing  rate  on  the  untreated  skin  must  be  achieved.  Due  to  the 
different aggressiveness of the mosquito species the following landing rates, corresponding to a 
worst-case landing pressure in the field (see Appendix 18: Moreno-Gómez, M., Bueno-Marí R., 
Drago A., et al.), must be achieved: 

•  Aedes spp. 20 landings/minute; 

•  Culex spp. 5 landings/minute; 

•  Anopheles spp. 5 landings/minute.  

The  density  and  number  of  animals  required  to  achieve  the  required  landing  rate  may  vary 
between mosquito species and between different studies. 

Environmental  conditions  must  be  specified  for  the  test  itself  (temperature,  humidity,  light 
intensity).  The  same  parameters  should  be  chosen  as  test  conditions  for  tropical  and  non-
tropical regions: temperature 27°C±2°C and relative humidity of 75%±5%. 

 
 
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5.6.5.9.2.2.1.2.1 Volunteers 

Efficacy data of at least 10 different volunteers, volunteers with different hairiness of the arms, 
different  genders;  age:  18-65  years,  should  be  collected  since  repellence/attractiveness  to 
mosquitoes varies considerably between human individuals. For CPT calculation valid data of at 
least 10 different volunteers must be used. Volunteers’ attractiveness is measured by exposing 
the  untreated  skin  to  caged  populations  (population  density,  cage  size  see  below)  of  host-
seeking, pathogen-free female mosquitoes, and the results must be presented in the report.  

To  avoid  unnecessary  blood  feeding,  the  volunteers  are  allowed  to  softly  shake  off  the 
mosquitoes  after  landing.  In  case  biting  activity  is  lower,  a  new  cage  with  fresh  mosquitoes 
should be used or additional mosquitoes could be added and reported. 

Volunteers need to be fully informed about the aim, the procedure, and the expected duration 
of a study. The study should be carried out in compliance with the national ethics regulation 38. 
They  also  need  to  be  informed  about  potential  side  effects  such  as  allergic  skin  reactions 
caused by the product. Their participation is voluntary and can be recalled at any time before 
and  during  the  study.  12  hours  before  and  during  testing,  volunteers  should  avoid  nicotine, 
alcohol,  fragrance (perfumes, body lotions, soap, etc.) and repellent products. Any side effect 
observed during the test should be mentioned in the test report. 

Prior to the application of the repellent, the skin is washed with fragrance-free soap and rinsed 
with  water.  The  hand  should  be  covered  with  a  glove  that  mosquitoes  cannot  bite  through 
during each exposure to the test insects. If the entire forearm is to be treated with repellent, 
the  skin  area  should  be  estimated  according  to  recommendations  of  the  WHO  (2009):  the 
mean circumference is  calculated by measuring the circumference at  the wrist and elbow and 
multiplied with the length of the arm (between wrist and elbow). 

Alternatively,  repellents  can  be  applied  to  a  defined  area  on  the  forearm,  e.g.  a  100  cm² 
window.  The  rest  of  the  arm  will  then  be  protected  from  bites  by  a  custom-made  sleeve 
(flexible  material  that  prevents  biting,  edges  are  not  treated  with  the  product;  contamination 
should  be  prevented).  The  use  of  a  defined  area  adds  to  standardisation  of  test  conditions; 
therefore,  the  use  of  a  sleeve  is  recommended.  Repellent  products  have  to  be  applied  at  the 
recommended label dose. 

5.6.5.9.2.2.1.2.2 Arm-in-Cage test 

A  simulated-use  test  with  the  following  criteria  could  be  conducted  to  determine  realistic  CPT 
values. 

Test cage: 

•  a  volume  between  27  L  and  64  L  can  be  used.  The  volume  should  be  at  least  27  L  to 

have enough space in the cage for the forearm; 

•  both a rectangular and a square shape are suitable; 

• 

the front side of the cage contains a sleeve for the introduction of the forearm and as a 
barrier for the mosquitoes to escape. 

Mosquito density: 

• 

the  density  and  number  of  animals  required  to  achieve  the  required  landing  rate  may 
vary between different trials. However, the following information may serve as a guide: 
A density between 1 female/840 cm³ and 1 female/640 cm³ should be chosen, e.g. 32-
42  females  in  27  L;  75-100  females  in  64  L.  The  number  of  mosquitoes  can  vary 
according to the mosquito species. 

Exposure period: 

• 

the treated skin should be exposed for 3 min in regular intervals. 

In each exposure period, prior to the efficacy test with the treated arm, the landing pressure of 
the pathogen-free test mosquitoes needs to be verified with the other, untreated arm. 

38  Declaration  of  Helsinki;  https://www.wma.net/policies-post/wma-declaration-of-helsinki-ethical-principles-for-
medical-research-involving-human-subjects 

 
 
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5.6.5.9.2.2.1.2.3 Arm-to-Cage test (see Appendix 18: Obermayr, U., et al.) 

Test  cages  are  usually  rectangular  and  have  a  volume  of  27  L,  e.g.  41x41x16  cm.  However, 
slightly larger cages can also be used, as long as the minimum mosquito landing pressure of 20 
landings per minute is met. The floor of the cage contains a test window, e.g. 60 cm² exposed 
skin  area  in  a  27  L  volume  cage  for  the  exposure  of  the  skin,  in  contrast  to  the  arm-in-cage 
test  the  arm  is  not  exposed  inside  the  test  cage.  Mosquito  landing  pressure  and  exposure 
period  should  follow  recommendations  made  in  section  5.6.5.9.2.2.1.2.2  “Arm-in-Cage  Test”. 
The treated skin area on the forearm is marked and should be larger than the test window in 
the floor of the cage to ensure that the exposed skin is entirely treated with repellent. Prior to 
the efficacy test of the treated arm, the landing pressure of the pathogen-free test mosquitoes 
needs to be verified with the other, untreated arm. 

5.6.5.9.2.2.1.2.4 Room test (see Appendix 18: Moreno-Gómez, M., Bueno-Marí R., Bowman, 
G.R., et al.) 

Tests should be conducted in free-flight, mosquito-proof rooms with a volume of at least 25 m³. 
For  day  active  mosquitoes  the  room  should  be  illuminated  by  external  lighting,  e.g.  150  W 
halogen fluorescent lighting, on the ceiling to eliminate shadows and white walls allow a better 
insect counting. All surfaces should be of a cleanable material, sealed and waterproof to avoid 
the accumulation of the product in the room. 

A sufficient number of female mosquitoes pre-selected for host-seeking behaviour are used per 
trial to achieve the required landing rate on the untreated arm. Test mosquitoes are allowed to 
acclimatize for 15 minutes in the test room before the volunteer enters the room. The landing 
rate  should be measured on the untreated arm of the  volunteer at  each test  interval. In case 
this minimum landing pressure is not achieved, additional mosquitoes can be added. Over the 
entire  test  an  average  of  the  required  landing  rate  must  be  achieved.  Due  to  the  natural 
behaviour,  variations  in  mosquito  activity  during  the  day  are  likely.  Therefore,  landing  rates 
below and above the required landing rate at the different test intervals are acceptable as long 
as the mean landing rate remains above the required landing rate during the whole observation 
period. 

Volunteers: Criteria described in section 5.6.5.9.2.2.1.2.1 must be  fulfilled. During the test,  a 
light beekeeper suit, gloves and white hospital booties should be used to protect the volunteer 
from  getting  bitten  (see  Figure  14).  The  untreated  arm  is  covered  with  the  suit,  whereas  the 
treated arm should be protected from bites and abrasion of the product by a “tube” of flexible 
material,  see  figure  1;  edges  are  not  treated  with  the  product;  contamination  should  be 
prevented. A handle inside the tube allows the volunteer to hold it. The tube is only used when 
the volunteer is in the cabin, the rest of the day the arm is exposed to the air. The volunteer 
enters  the  cabin  with  both  forearms  covered  and  walks  through  the  cabin  (approximately  2 
minutes)  until  discovered  by  the  mosquitoes.  Then  the  volunteer  stops  walking,  exposes  the 
untreated  arm  and  counts  the  number  of  landings,  e.g.  for  Aedes  albopictus  for  an  exposure 
period of 3 minutes. The exposure period should be the same during the study and should be 
adapted to the mosquito species. After the landing rate has been determined as sufficient, the 
volunteer  covers  the  untreated  arm  and  immediately  exposes  the  treated  arm.  The  exposure 
period starts once the treated arm is fully exposed. 

 
 
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Figure  14:  Room  test  volunteer  wearing  a  light  beekeeper  suit,  gloves  and  white  hospital  booties  and  one  arm  is 
protected by a “tube” of flexible material. 

After  each  efficacy  test,  the  room  needs  to  be  cleaned  thoroughly  before  the  next  test  is 
initiated. 

5.6.5.9.2.2.1.2.5 Efficacy assessment 

Mosquito strain, age and larval diet should be recorded. 

While testing for repellence, an endpoint for  failure of repellence for subjects treated with the 
recommended label dose should be  selected. Efficacy failure in a test  to determine  CPT is the 
time  from  application  of  a  repellent  until  efficacy  failure  by  a  confirmed  event  (definition  see 
Introduction, section 5.6.5.1.5). Repellent efficacy should be based on the median or mean CPT 
(CPT calculation see Introduction, section 5.6.5.1.5).  

The claimed CPT for a general claim should correspond to the shortest CPT (mean or median) 
among  all  tested  species  and  should  be  stated  on  the  label  and  SPC.  For  specific  claims  the 
mean or median for each species respectively may be stated separately on the label and SPC. 

Proof  of  non-insecticidal  efficacy,  if  it  cannot  be  waived  (for  details  see  Introduction,  section 
5.6.5.1.3.8):  After  the  test  or  in  a  separate  test,  test  mosquitoes  are  provided  with  a  10% 
sugar solution and maintained within the test cage or test room to observe whether insecticidal 
effects could be caused by contact/exposure to the test repellent product. The mortality of the 
insects should be monitored at the end of the test. 

5.6.5.9.2.2.1.3 Field test 

In order to eliminate the risk of disease transmission to human volunteers in field settings, field 
trials are not required for authorisation of products applied on human skin. Pre-existing studies 
may  be  submitted  as  additional  information  because  field  trials  are  not  assessed  as  key 
studies.  

If  field  trials  are  conducted,  they  must  take  place  in  an  area  with  an  appropriate  mosquito 
biting  pressure  and  at  a  time  when  the  relevant  mosquito  species  are  active.  The  required 
biting pressure will depend on the species: 

•  Aedes spp.: at least 1 probe or bite/min is required, 

•  Anopheles spp.: at least 1 probe or bite/min is required, 

•  Culex spp. can be less active. The biting pressure chosen for test conduction should be 

justified in the test report. 

 
  
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These  tests  should  preferably  take  place  in  Europe  or  other  relevant  regions  according  to  the 
claims,  e.g.  tropical  regions.  Where  tests  in  Europe  are  not  possible  the  conditions  and 
mosquito species must be confirmed, and their relevance justified. 

Field  trials  should  follow  the  WHO  or  EPA  protocol,  see  Appendix  18.  In-house  protocols  are 
also acceptable; any deviations should be justified. 

Field  tests  allow  the  evaluation  of  topical  repellents  against  wild  mosquito  populations  under 
realistic  conditions  at  the  recommended  label  dose.  In  contrast  to  simulated-use  tests,  they 
are,  however,  subjected  to  greater  variation  regarding  biting  activity,  mosquito  species, 
population density, temperature, humidity, light conditions, etc. Since these factors cannot be 
controlled,  field  tests  should  be  conducted  in  a  field  site  in  an  environmentally  distinctive 
habitat, e.g. forest, wetland, where the predominant mosquito species differ. 

Volunteers: Criteria described in section 5.6.5.9.2.2.1.2.1 must be fulfilled. Volunteers trained 
on the use of an aspirator can serve as their own control on the other, untreated, arm or leg. 
Alternatively, an equal number of untreated volunteers could be included in the field trial, as a 
negative control. 

The  forearm  (between  wrist  and  elbow)  or  lower  leg  (between  knee  and  ankle)  serves  as  the 
treatment  area.  The  skin  area  should  be  measured  and  treated  as  described  in  section 
5.6.5.9.2.2.1.2.1.  Product  application  must  follow  the  claimed  instruction  of  use.  The  treated 
skin  will  be  exposed  in  regular  intervals  for  not  less  than  3  minutes.  The  frequency  of 
assessments will be not less than every hour until CPT ends (occurrence of the first confirmed 
bite,  see  Introduction,  section  5.6.5.1.5).  Exposure  periods  should  be  synchronized  with  the 
time  when  the  relevant  species  are  abundant.  The  treatment  of  arms  or  legs  should  also  be 
consistent  with  the  feeding  preference  of  the  target  species.  Time  of  day  at  which  volunteers 
are  treated  and  at  which  exposure  started  and  ended  should  be  reported  along  with  the 
weather  conditions  (temperature,  humidity,  precipitation,  wind  speed,  light  intensity, 
cloudiness).  Mosquitoes  that  probe  or  bite  during  an  efficacy  test  can  be  collected,  where 
possible,  for  later  identification.  Before  the  study  is  conducted,  the  presence  of  the  relevant 
mosquito species has to be verified. This should be documented in the study report. 

5.6.5.9.2.2.2 Repellents applied on clothing both for humans or animals 

For  product  authorisation  purposes  the  efficacy  of  repellent  products  applied  on  clothing  for 
humans  should  be  proven  in  a  simulated-use  test  according  to  the  instructions  for  use 
(description see 5.6.5.9.2.2.2.1) 

CPT  values  must  be  determined  in  a  simulated-use  test.  As  field  studies  are  not  assessed  as 
key studies, data from the field studies cannot be used to determine CPT values (definition and 
calculation see Introduction 5.6.5.1.5). 

In order to eliminate the risk of disease transmission to human volunteers in field settings, field 
trials with repellents against mosquitoes are not required for authorisation of products applied 
on clothing to protect  human skin (for details  see  Introduction,  section 5.6.5.1.3.3.3). If  field 
trials  are  conducted,  they  must  take  place  in  an  area  with  an  appropriate  mosquito  biting 
pressure (see 5.6.5.9.2.2.1.3) and at a time when the relevant mosquito species are abundant. 
These  tests  should  preferably  take  place  in  Europe  or  other  relevant  regions  according  to  the 
claims  (e.g.  tropical  regions).  Where  tests  in  Europe  are  not  possible  the  conditions  and 
mosquito species must be confirmed, and their relevance justified. 

For  product  authorisation  purposes  the  efficacy  of  repellent  products  applied  on  clothing  for 
animals should be proven in: 

•  a  simulated-use  test  according  to  the 

instructions 

for  use  (description  see 

5.6.5.9.2.2.3.2), or 

•  a field test according to the instructions for use.  

The repellents used for the test should be identical to the product to be marketed. 

Proposed  label  claims  regarding  the  protection  of  a  specific  type  of  fabric  must  be  simulated, 
i.e. the same type of fabric has to be used in the simulate-use test. 

Proposed label claims regarding the performance of the product must be simulated in the study 
(for details see Introduction, section 5.6.5.1.3.5). 

 
 
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5.6.5.9.2.2.2.1 Simulated-use test 

The efficacy of products intended for use on clothes or fabrics for humans must be proven in a 
similar test as described in section 5.6.5.9.2.2.1.2 by covering the test arm with a treated cloth 
and the control arm with untreated cloth, or simulating the normal use of the treated fabric in 
case  of  products  to  protect  animals  (see  5.6.5.9.2.2.3.2),  depending  on  the  claim.  The  fabric 
used has to be representative of the fabrics commercially used, i.e. thick uniform cotton if the 
field of use is military clothing, etc. 

Proof  of  non-insecticidal  efficacy,  if  it  cannot  be  waived  (for  details  see  General  Introduction, 
chapter  5.6.5.1.3.8):  After  the  test,  test  mosquitoes  are  provided  with  a  10%  sugar  solution 
and maintained within the test cage or test room to observe whether insecticidal effects could 
be  caused  by  contact/exposure  to  the  test  repellent  product.  The  mortality  of  the  insects 
should be monitored at the end of the test. 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  substrates/surfaces  or  repellent  product  (temperature,  humidity,  photoperiod, 
ventilation). 

If  outdoor  use  is  claimed,  the  treated  surface/substrate  or  repellent  product  and  control 
surfaces/substrates  have  to  be  kept  outside  to  ensure  adequate  weathering  and  the  outdoor 
conditions must be specified (for detailed information regarding the requirements see General 
Introduction, chapter 5.6.5.1.4.1.3). 

The  efficacy data should be  relevant to prove  the submitted  claims. Therefore, the  efficacy of 
biocidal products that are limited to a body area e.g. collars for animals must be proven for the 
whole  test  individual  that  should  be  protected.  If  only  specific  body  areas  intend  to  be 
protected, e.g. ears, udders, etc. then the efficacy for the claimed area must be proven. 

For specific claims (prevention of bites through/prevention of bites next to the treated clothes), 
relevant tests have to be submitted. 

5.6.5.9.2.2.3 Products intended for use as topical repellent for animals  

For product authorisation purposes the efficacy of repellent products applied on animals should 
be proven in: 

•  a  simulated-use  test  according  to  the  instructions  for  use  (test  design  example  see 

5.6.5.9.2.2.3.2), or 

•  a field trial (test design example see 5.6.5.9.2.2.3.3). 

Topical  repellents  for  animals  should  be  tested  against  the  targeted  mosquito  species  and  on 
the animal, e.g. dog, horse, etc., that shall be protected. Dogs should be used as a substitute 
for cats, as testing with cats can imply certain animal welfare issues. 

Proposed claims regarding the performance of the product must be simulated in the study. For 
the claim “unaffected  by washing”, the label and the SPC must indicate how often the animal 
can  be  washed  without  reducing  the  efficacy  of  the  biocidal  product.  In  the  efficacy  test,  the 
test  individuals  should  be  repeatedly  washed  with  a  non-insecticidal,  fragrance-free  shampoo 
during the trial according to the number of wash cycles indicated. 

The repellents used for the test should be identical to the product to be marketed. 

Test animals: It is recommended to include at least 10 animals per treatment/control group of 
different  breed  and  sex,  i.e.  for  testing  on  entire  animals  at  least  10  treated  and  10  control 
individuals  are  necessary  since  repellence/attractiveness  to  mosquitoes  varies  considerably 
between animal individuals. 

Depending  on  the  animal  species,  several  factors  e.g.  hair  length,  the  thickness  of  the  coat, 
self-grooming, etc. might impact the efficacy. These factors should be taken into account in the 
demonstration  of  the  efficacy.  The  two  groups  should  be  kept  separately,  to  avoid  contact  of 
the control animals with the treated fur. 

The  product  should  be  applied  according  to  the  claim.  Claimed  application  rates  should  take 
into account the type and weight of the animals.  

It  is  necessary  that  animals  come  from  suitable,  e.g.  non-smoking  households.  Every  side 
effect  observed  during  the  test  should  be  mentioned  in  the  test  report.  24  hours  before  and 

 
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during  testing,  animals  should  not  be  treated  with  fragrance  (perfumes,  soap,  etc.).  The  test 
animal  should  not  be  protected  by  any  previous  repellent  and/or  insecticidal  residuals  caused 
by  previous  treatment.  Concerning  endo-antiparasitic  agents  this  is  acceptable,  but  details 
(product,  dose,  application  rate,  application  method,  date  of  the  last  treatment,  and  residual 
efficacy)  must  be  given  in  the  report  and  the  controls  and  the  treated  groups  must  have  the 
same  status.  A  safety  margin  for  externally  applied  anthelmintics  is  4  weeks  after  the  end  of 
the  last  application.  The  history  of  the  treated  and  the  control  groups  should  be  comparable. 
The status of the animals should be specified and only animals for which an insecticidal effect 
can  be  excluded  should  be  included  in  the  study.  Due  to  feasibility  and  cost-effectiveness,  it 
would  be  possible  to  use  10  animals  one  week  (5  each  in  the  treatment  and  control  groups) 
and  then  use  the  same  individuals  again  in  the  second  week,  but  swap  groups.  Each  animal 
would  then  act  as  its  own  control.  However,  any  residual  activity  of  the  product  has  to  be 
excluded. 

The investigator is reminded that the validity of the results is directly related to the degree of 
variability in the test. Increasing the number of test animals could increase the reliability of the 
test results. 

5.6.5.9.2.2.3.1 Laboratory test  

A choice test similar to the tests described in section 5.6.5.9.2.2.1.1 could be used. 

5.6.5.9.2.2.3.2 Simulated-use test  

In the simulated-use tests, the product is applied according to the instructions for use. If the 
product  is  acting  at  very  close  range  it  may  be  possible  to  make  the  evaluation  by  treating 
parts  of the host, like  one treated  side  of the  neck  compared with the equivalent area  on the 
other,  untreated,  side  of  the  neck.  The  advantage  of  such  a  setup  is  the  neutralisation  of 
individual  host  differences  in  mosquito-attraction.  The  control  body  part  should  resemble  the 
treated body part, e.g. front-half vs back-half or left side vs right side of the animal. In case of 
a restricted application, e.g. collar, a spot-on product with a claim of entire body protection or 
for products acting in a wider range over the entire body the test should permit to validate the 
efficacy of this kind of application by testing the entire animal. In this case, untreated animals 
must be used as a control. 

The detailed measurements will depend on the size of the animal, and the following dimensions 
adapted from the simulated-use test on humans should be used only as guidance. 

Test  animals:  The  attractiveness  of  the  animal  should  be  validated  before  the  trial.  Animal’s 
attractiveness  can be  measured by  exposing  the untreated  skin to caged  populations  of host-
seeking  female  mosquitoes.  Repellent  products  have  to  be  applied  at  the  recommended  dose 
and  according  to  the  claimed  mode  of  application.  Claimed  application  rates  should  take  into 
account  the  type  and  weight  of  the  animals.  The  animals  tested  should  be  representative  for 
the claims. 

The parameter for determining the number of mosquitoes in the cages is the landing rate. For 
details see 5.6.5.9.2.2.1.2. 

Mosquitoes  are  attached  to  the  treated  skin  in  screened  cages  to  force  them  on  the  defined 
treated area and landing and biting behaviour is documented in regular intervals, see Appendix 
18: EPA and EMA Guidelines. 

Alternatively,  test  animals  could  be  placed  inside  a  screened  cage  with  host-seeking  female 
mosquitoes. Untreated body parts should be covered to prevent excessive biting. 

Environmental  conditions  must  be  specified  for  the  test  itself  (temperature,  humidity,  light 
intensity).  The  temperature  would  be  expected  to  fall  in  the  range  of  27°C±2°C  and  relative 
humidity of 75%±5%.  

Proof  of  non-insecticidal  efficacy,  if  it  cannot  be  waived  (for  detail  see  Introduction,  section 
5.6.5.1.3.8):  After  the  trial,  test  mosquitoes  are  provided  with  a  10%  sugar  solution  and 
maintained  within  the  test  cage  to  observe  whether  insecticidal  effects  could  be  caused  by 
contact/exposure  to  the  test  repellent  product.  The  mortality  of  the  insects  should  be 
monitored at the end of the test. 

Efficacy  assessment  should  be  based  on  CPT,  definition  and  calculation,  see  Introduction, 
section 5.6.5.1.5. 

 
 
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The claimed CPT for a general claim should correspond to the shortest CPT (median or mean) 
among  all  tested  species  and  should  be  stated  on  the  label  and  SPC.  For  specific  claims,  the 
mean or median for each species respectively may be stated separately on the label and SPC. 

Authorisation of products applied with a dose independent of animal size can be granted up to 
the bodyweight of the heaviest test animal. 

5.6.5.9.2.2.3.3 Field test  

For PT19 products which are applied on animals, no common protocols are currently available. 
However, several guidelines for the evaluation of ectoparasiticides are available, see Appendix 
18. These guidelines can be adapted for the demonstration of the efficacy of repellent products 
to be applied on animals. 

5.6.5.9.2.2.4 Products intended for use as general surface treatment  

The efficacy of general surface treatment products should be proven either in: 

•  a simulated-use test according to the instructions for use, or 

•  a field test according to the instructions for use and additionally a laboratory trial testing 

the required different surfaces. 

Products applied onto surfaces may act either by evaporation or on the surface itself. 

For products applied on surfaces, two porous and one non-porous surface should be used, e.g. 
ceramic  tile,  plywood,  painted  plywood,  stainless  steel,  concrete,  for  a  general  label  claim  as 
“surface treatment”. The efficacy of each surface should be proven in a separate test, i.e. three 
tests for three different surfaces. For authorisation of a product to be used on a specific type of 
surface the efficacy for only this specific surface should be assessed. 

Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative  control) 
should be performed.  

Environmental  conditions  must  be  specified  for  the  test  itself,  and  during  the  storage  of  the 
treated  substrates/surfaces  or  repellent  product  (temperature,  humidity,  photoperiod, 
ventilation). 

For  residual  efficacy,  mosquitoes  are  exposed  to  the  product  at  several  time  intervals  after 
application (including the end of the claimed period). 

Proposed  label  claims  regarding  the  performance  of  the  product  should  be  simulated  in  the 
study.  For  example,  for  the  claim  “unaffected  by  cleaning/vacuuming”,  the  surface  should  be 
repeatedly cleaned during the trial (for details see Introduction, section 5.6.5.1.3).  

The repellents used for the test should be identical to the product to be marketed. 

If  outdoor  use  is  claimed,  the  treated  surface/substrate  or  repellent  product  and  control 
surfaces/substrates  have  to  be  kept  outside  to  ensure  adequate  weathering  and  the  outdoor 
conditions must be specified (for details see Introduction, section 5.6.5.1.4.1.3). 

The repellents used for the test should be identical to the product to be marketed. 

Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see  Introduction, 
section 5.6.5.1.3.8). 

5.6.5.9.2.2.5 Products intended for use as spatial repellents 

For product authorisation purposes the efficacy of spatial repellent products, including repellent 
emanators  (electronic  vaporizers,  candles,  wristbands,  passive  vaporizers  for  volatile 
pyrethroids, coils) should be proven in: 

•  a  simulated-use  test  according  to  the  instructions  for  use  (test  design  example  see 

5.6.5.9.2.2.5.1), or 

•  a  field  test  according  to  the  instructions  for  use  (test  design  example  see 

5.6.5.9.2.2.5.2). 

When the label claim says that the product should be used in ventilated rooms the opening of 
windows and doors should be simulated in the test. 

 
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The  room  size  stated  in  the  SPC  and  on  the  label  must  be  used  in  the  efficacy  trial.  For  the 
extrapolation to larger room sizes than proven in the efficacy test a justification is necessary. 

If a claim  states that the product prevents insects  from landing/probing, a simulated-use test 
proving the landing/probing inhibition with a volunteer in the room is necessary. 

If a claim states that the product prevents insects from entering a space, a simulated-use test 
proving the entry reduction is necessary. 

In  case  of  a  specific  claim  regarding  the  use  in  specific  climate  conditions,  e.g.  high 
temperature or tropical conditions, the efficacy must be proven under these conditions. 

Environmental  conditions  must  be  specified  for  the  test  itself,  and  during  the  storage  of  the 
treated  substrates/surfaces  or  repellent  product  (temperature,  humidity,  photoperiod, 
ventilation). 

If  outdoor  use  is  claimed,  the  test  should  be  performed  outdoor  and  the  treated 
surface/substrate or repellent product and control surfaces/substrates have to be kept outside 
to  ensure  adequate  weathering  and  the  outdoor  conditions  must  be  specified  (for  details  see 
Introduction, section 5.6.5.1.4.1.3). 

The repellents used for the test should be identical to the product to be marketed. 

5.6.5.9.2.2.5.1 Simulated-use tests 

Conducting simulated-use tests  ensures that the mosquitoes are pathogen-free, that a known 
number of mosquitoes of a known age is used and that the distance between the point at which 
the  mosquitoes  are  released  and  the  source  of  the  chemical  stimulus  can  be  defined,  which 
allows the estimation of the protective area (especially important in outdoor evaluation). Other 
(5.6.5.9.2.2.5.1.1 
test  designs 
Landing/probing  inhibition  test;  5.6.5.9.2.2.5.1.2  Reduction  of  entry  into  an  area)  can  be 
accepted if the protocol is scientifically valid.  

following  examples  adapted 

from  WHO 

than 

the 

Mandatory requirements: 

•  Replicates:  A  minimum  of  5  independent  replicates  with  different  volunteers  (each 

treatment and negative control) should be performed. 

•  A  sufficient  number  of  female  mosquitoes  pre-selected  for  host-seeking  behaviour  are 
used per trial to achieve a minimum  landing rate. The landing rate is determined after 
the host is discovered  by the mosquitoes (approximately after 2 minutes).  The density 
and  number  of  mosquitoes  required  to  achieve  this  rate  may  vary  between  different 
trials.  Over  the  entire  test  an  average  of  the  required  landing  rate  must  be  achieved. 
Due  to  the  natural  behaviour,  variations  in  mosquito  activity  during  the  day  are  likely. 
Therefore, landing rates below and above the required landing rate at the different test 
intervals  are  acceptable  as  long  as  the  mean  landing  rate  remains  above  the  required 
landing rate during the whole observation period. 

•  Required  minimum  landing  rates:  Due  to  the  different  aggressiveness  of  the  mosquito 
species  the  following  landing  rates,  corresponding  to  a  worst-case  landing  pressure  in 
the field, must be achieved: 

-  Aedes spp. 20 landings/minute; 

-  Culex spp. 1 landing/minute; 

-  Anopheles spp. 1 landing/minute.  

•  For  residual  efficacy,  several  trials  should  be  conducted,  in  which  the  mosquitoes  are 
exposed  to  the  product  at  several  time  intervals  after  application  (including  the  end  of 
the claimed period). 

•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8). 

5.6.5.9.2.2.5.1.1 Landing/probing inhibition test 

The  following  design  example  for  a  simulated-use  test  was  adapted  from  WHO,  see  Appendix 
18.  Other  test  designs  than  the  following  examples  can  be  accepted  if  the  protocol  is 
scientifically valid. 

 
 
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Products that are intended to be used indoors:  

Tests should be conducted in free-flight rooms (mosquito-proof and well ventilated by opening 
the door and/or window for a minimum of 1 air exchange/hour, see Appendix 18: te Biesebeek, 
J.D., et al.) that measure at least 30 m³. The test product is installed and activated according 
to  the  label  claim.  Test  mosquitoes  are  allowed  to  acclimatize  for  1  hour  under  conditions 
similar  to  the  test  room  and  then  released  into  the  test  room.  The  trained  volunteer  or  a 
trained  investigator  documents  landing  and  probing  behaviour.  After  each  efficacy  trial,  the 
room  needs  to  be  cleaned  thoroughly  before  the  next  trial  is  initiated.  The  efficacy  of  the 
repellent product is compared to control tests without product or with the pre-treatment. 

Products that are intended to be used outdoors:  

Tests  can  be  performed  in  experimental  open  areas  of  a  minimum  of  30  m³  inside  screened 
enclosures. Ideally, several areas  should be available to allow simultaneous  comparisons. The 
product is installed according to the label claim and a volunteer is positioned at the  centre  of 
the test area to perform human landing collections. Female mosquitoes are released outside of 
the  test  area.  Landing  and  probing  behaviour  is  documented  for  defined  periods  of  time  that 
depend  on  the  product  label  specifications  Tests  should  be  conducted  during  the  main  biting 
activity  of  the  tested  species.  Temperature,  humidity,  wind  speed  and  direction  should  be 
recorded for the duration of each replicate. 

Efficacy assessment: Percent landing/probing inhibition is based on the number of mosquitoes 
landing/probing in the control subtracting from this the number of mosquitoes landing/probing 
in the treated room and divided by the number of mosquitoes landing/probing in the control. 

Proof  of  non-insecticidal  efficacy:  In  the  test  chamber  a  cage  (most  appropriate  consisting  of 
mosquito  net  ensuring  a  good  ventilation)  containing  mosquitoes  provided  with  a  10%  sugar 
solution  is  maintained  for  1  hour  during  the  test  to  observe  whether  insecticidal  effects  could 
be  caused  by  exposure  to  the  test  repellent  product.  Mortality  of  the  insects  should  be 
monitored at the end of the test. 

5.6.5.9.2.2.5.1.2 Reduction of entry into an area 

A spatial repellent is applied to protect an area of X m². 

The  following  design  example  for  a  simulated-use  test  was  adapted  from  WHO,  see  Appendix 
18.  Other  test  designs  than  the  following  examples  can  be  accepted  if  the  protocol  is 
scientifically valid. 

Products that are intended to be used indoors:  

Tests  are  conducted  in  two  adjacent  rooms  that  allow  mosquito  movement  from  one  room  to 
the other,  e.g. through a window  opening.  The test product is installed according to the label 
claim  in  one  room,  while  mosquitoes  are  released  in  the  other.  The  number  of  entering 
mosquitoes is documented for 10-15 minutes every hour by one trained volunteer or a trained 
investigator  until  the  end  of  the  claimed  period.  Between  these  observation  periods,  the 
connecting window should be kept closed, in order to avoid accumulation of the product in both 
rooms and to maintain a gradient of the active substance. 

Products that are intended to be used outdoors:  

Test  should  be  performed  in  outdoor  screened  cages  separated  into  two  compartments  that 
allow mosquito movement from one room to the other. The test product is installed according 
to the label claim in one compartment, while mosquitoes are released in the other. A volunteer 
sits down in the compartment without the mosquitoes at a set distance to the treatment device 
or  control,  according  to  the  label  claim,  if  applicable.  The  number  of  entering  mosquitoes  is 
documented for certain observation periods, e.g. at hour 1, hour 2 and hour 3. Tests should be 
conducted  during  the  main  biting  activity  of  the  tested  species.  The  number  of  entering 
mosquitoes  is  documented  for  defined  periods  of  time  that  depend  on  the  product  label 
specifications.  Temperature,  humidity,  wind  speed  and  direction  should  be  recorded  for  the 
duration of each replicate. 

Efficacy assessment: Percent reduction of entry is based on the number of mosquitoes entering 
the  test  area  in  the  control  subtracting  from  this  the  number  of  mosquitoes  entering  the 
treated test area/room and divided by the number of mosquitoes entering the test area in the 
control. 

 
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Proof  of  non-insecticidal  efficacy:  In  the  test  chamber  a  cage  (most  appropriate  consisting  of 
mosquito  net  ensuring  a  good  ventilation)  containing  mosquitoes  provided  with  a  10%  sugar 
solution  is  maintained  for  1  hour  during  the  test  to  observe  whether  insecticidal  effects  could 
be  caused  by  exposure  to  the  test  repellent  product.  Mortality  of  the  adult  insects  should  be 
monitored at the end of the test. 

5.6.5.9.2.2.5.2 Field trial 

The repellent effect should be demonstrated for each mosquito species claimed. 

The product should be applied according to the claim. 

For  indoor  evaluations,  several  houses  should  be  used,  when  feasible.  Houses  should  be  well 
described,  especially  with  regard  to  the  conditions  relevant  to  product  efficacy,  including 
estimates  of  indoor  volume  and  air  ventilation,  e.g.  sealed  or  gapped  walls,  number  of 
windows,  doors,  or  eave  area.  Houses  are  randomized  to  receive  either  active  (formulated 
spatial  repellent)  or  control  (placebo;  inert  ingredients  alone)  treatment  during  the  trial. 
Collectors should be ‘blinded’ to treatment allocation. 

In  outdoor  evaluations,  spatial  repellent  product  or  control  should  be  allocated  randomly  to 
comparable outdoor spaces with human exposure.  

Replicates:  A  minimum  of  3  independent  replicates,  i.e.  3  different  houses,  (each  treatment 
and negative control) should be performed.  

Efficacy  assessment:  Percent  landing/probing  inhibition  or  reduction  of  entry  is  based  on  the 
number of mosquitoes landing/probing/entering in the control subtracting from this the number 
of  mosquitoes  landing/probing/entering  in  the  treated  room  and  divided  by  the  number  of 
mosquitoes landing/probing/entering in the control. 

5.6.5.9.2.2.6 Attractants without PT18 active substances  

For  product  authorisation  purposes  the  efficacy  of  attractants  in  traps  without  an  active 
substance according to PT18 should be proven in: 

•  a  simulated-use  test  according  to  the  instruction  for  use  (test  design  example  see 

5.6.5.9.2.2.6.2), or 

•  a 

field  test  according  to  the 

instruction 

for  use  (test  design  example  see 

5.6.5.9.2.2.6.3). 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  storage  of  the 
attractant product (temperature, humidity, photoperiod, ventilation).  

If  outdoor  use  is  claimed,  the  test  should  be  performed  outdoors  and  the  attractant  product 
and control have to be kept outside to ensure adequate weathering and the outdoor conditions 
must be specified (for details see Introduction, section 5.6.5.1.4.1.3). 

The attractants, e.g. the traps, used for the test should be as similar as possible to the product 
to be marketed (for details see Introduction, section 5.6.5.1.3.7).  

5.6.5.9.2.2.6.1 Laboratory test 

An olfactometer test similar to design described under 5.6.5.9.2.2.1.1.1 can be used. 

5.6.5.9.2.2.6.2 Simulated-use test 

As no test guidelines are currently available, any inhouse protocol may be used, if the protocol 
is scientifically valid. A test set-up similar to the ones described above (5.6.5.9.2.2.5.1) should 
be used where suitable. Conditions should mimic the claimed use. 

Replicates: Tests should be performed with at least 5 replicates, and 5 negative controls should 
be used. 

The number  of mosquitoes released into the  room  or  enclosure needs to be recorded and put 
into relation to the captured mosquitoes. 

5.6.5.9.2.2.6.3 Field trial  

In the field tests, mosquito attractants that are intended to be used with trapping systems  or 
within  other  “lure  and  kill”  products  should  be  evaluated  against  the  claimed  target  species. 
Attractants  should  be  tested  in  combination  with  a  system  that  allows  the  documentation  of 

 
 
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captured  specimen,  e.g.  a  commercial  mosquito  trapping  device  (see  Appendix  18: 
Vythilingam, I. et al., Kline, D.L., et al., Okumu, F., et al.). The location of the attractant within 
the trap or system needs to be documented. 

Any attractant should be tested in a comparison between a trapping device equipped with  the 
attractant to an untreated  reference device of  the same model, in a 2x2 Latin square design. 
The attractant must show a statistically significant increase in catch rates, as compared to the 
control trap. 

Field tests need to be conducted in a biotope with a natural occurrence of the target mosquito 
species. 

The  distance  between  test  locations  depends  on  the  claim,  on  the  species  and  the 
environmental conditions  (suggested distance:  30-50 m). Test locations should be far  enough 
apart to ensure that interactions between traps (attractants) do not occur, but near enough to 
experience  similar  weather,  biotope  and  comparable  mosquito  densities.  Traps  are  installed 
according to the manufacturer's recommendations. Trap operation hours depend on the target 
species main activity: all traps should be switched on at the same time and be operated during 
peak activity hours of the target species, e.g. between dusk and dawn. Alternatively, traps can 
be  operated for 24 hours.  Operation hours need to be the same for all treatments  during the 
entire  experiment.  At  the  end  of  a  catch  period,  mosquito  collections  are  removed  from  the 
trap and stored for later counting and identification. 

Any bycatch of non-target insects has to be recorded and identified at least to order, preferably 
to the family level. 

5.6.5.9.2.2.7 Attractants in PT18 bait products 

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for  bait 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods). 

5.6.5.9.3 Assessment of authorisation  

5.6.5.9.3.1 Norms and criteria  

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for mosquitoes in the following way: 

Products intended for use as repellent for human or animal skin, clothing: 

Non-insecticidal  efficacy  has  to  be  proven  in  a  simulated-use  test  if  it  cannot  be  waived  (for 
details see Introduction, section 5.6.5.1.3.8). 

The required results for the different trials are: 

Laboratory tests (not required for product authorisation): 

•  100% repellency for the claimed time period. 

Simulated-use tests and field trials: 

•  during the claimed protection period complete protection should be proven expressed as 

mean or median CPT; 

•  CPT should correspond to the shortest one among the tested species; for specific claims 

the mean or median for each species can be stated on the label). 

Products intended for use as general surface and spatial repellents: 

Non-insecticidal efficacy has to be proven in a simulated-use test (for details see Introduction, 
section 5.6.5.1.3.8). 

The required results for the different trials are: 

Laboratory tests (not required for product authorisation):  

•  95% repellency for the claimed time period. 

Simulated-use tests: 

•  ≥ 80% percent landing/probing inhibition within the test period according to the claim, 

from the beginning and until the end of the claimed efficacy period, or 

 
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•  ≥  80%  reduction  of  entry  within  the  test  period  according  to  the  claim,  from  the 

beginning and until the end of the claimed efficacy period. 

Field trials: 

•  ≥ 80% percent landing/probing inhibition  within the test period according to the claim, 

from the beginning and until the end of the claimed efficacy period, or 

•  ≥  80%  reduction  of  entry  within  the  test  period  according  to  the  claim,  from  the 

beginning and until the end of the claimed efficacy period. 

Attractants without PT18 active substances: 

The required results for the different trials are: 

•  at least a ratio of 4:1 of test individuals trapped in the trap with attractant compared to 
the  control  trap  within  the  test  period  (or  according  to  the  claim),  from  the  beginning 
and until the end of the claimed efficacy period.  

Laboratory tests (not required for product authorisation), simulated-use test: 

•  ≥  80%  attraction  (number  of  released  mosquitoes  in  relation  to  captured  animals) 
within the test period according to the claim, from the beginning and until the end of the 
claimed efficacy period. 

Field trials: 

•  ≥ 80% attraction within the test period according to the claim, from the beginning and 

until the end of the claimed efficacy period. 

5.6.5.10 Stable flies indoor 

Evaluation of Stomoxys calcitrans outdoor is part of the description for flies on grazing cattle, 
horses and other livestock. 

5.6.5.10.1 Introduction 

The  stable  fly  (Stomoxys  calcitrans)  is  a  synanthropic  fly  of  veterinary  importance  found 
worldwide.  It  is  an  obligate  blood-feeder  which  prefers  to  feed  on  cattle,  horses  and  pigs.  In 
the  absence  of  the  primary  hosts,  they  may  bite  humans  as  well  as  dogs.  The  bite  is  very 
painful and provokes considerable unrest, often causing defensive behaviour of the hosts when 
the host tries to get free of the stable flies. Horses and cattle, which are heavily infested may 
even  become  anaemic.  It  has  been  estimated  that  a  decline  of  up  to  25%  in  milk  production 
can  be  attributed  to  stable  flies  dependent  on  the  level  of  infestation.  Additionally,  the  stable 
fly may act as a mechanical vector of various pathogens. 

5.6.5.10.1.1  Biology 

The  stable  fly,  Stomoxys  calcitrans  L.,  (6-9  mm)  is  a  medium  size  brown-greyish  fly  with  an 
appearance  not  much  different  from  a  house  fly  (Musca  domestica),  both  belonging  to  the 
family Muscidae. The two species may, on a superficial level, be confused with one another, as 
they are both very common in stables, cowsheds and pigsties. The house fly is a sucking fly not 
capable  of  penetrating  the  skin  while  the  stable  fly  is  a  biting  fly  belonging  to  the  subfamily 
Stomoxyinae  (genus:  Stomoxys)  and  without  a  doubt  distinguishable  from  house  flies  by  its 
long, slender prominent, forward-directed, pointed proboscis. 

The  stable  fly  is  a  cosmopolitan  species.  In  the  northern  parts  of  Europe,  the  stable  fly  goes 
through  its  complete  life-cycle  in  stables  with  livestock,  especially  cattle  and  pigs.  On  warm 
days,  or  in  warmer  southerly  climates,  flies  may  migrate  between  hosts  in  nearby  fields;  the 
breeding  and  mating  take  place  on  the  farm.  Both  sexes  are  blood-feeding,  needing  frequent 
blood  meals  and  usually  take  one  blood  meal  per  day.  Stable  flies  favour  blood  from  cattle, 
they  also  appreciate  blood  from  horses  and  pigs  as  well  and  if  forced  they  may  bite  other 
warm-blooded  animals.  Cattle  and  horses  are  in  general  attacked  on  the  lower  parts  of  the 
body and on the extremities, especially the forelimbs. Pigs are more often attacked behind the 
ears. During the dark hours, the stable flies are resting high in buildings, on ceilings and upper 
parts of the walls. Their main blood-seeking activity is highest between 10 am and 4 pm, but 
this  is  not  very  strict,  and  they  may  bite  any  time  in  the  light  hours  if  the  conditions  are 
suitable. 

 
 
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5.6.5.10.2 Dossier requirements 

Dossier requirements are stated in the General Introduction (see section 5.6.5.1.3). 

5.6.5.10.2.1 Test species  

Tests  with  laboratory  colonies  of  stable  flies  as  well  as  with  field  collected  specimens  are 
applicable. In field experiments it must be clearly described how the test method ensured that 
only stable flies were counted in the experiment. 

Products intended for use as repellents on domestic animals or as spatial repellents should be 
tested  with  the  claimed  host,  e.g.  cattle,  horse  or  sheep.  A  general  host  claim  is  not 
acceptable. 

Repellent testing should be performed with stable flies. It is not acceptable to use results from 
other fly species to support a claim for stable flies. 

In  case  of  specific  claims,  e.g.  effective  at  high  temperature,  in  contact  with  water,  host 
sweating, etc., efficacy must be demonstrated in the relevant situations. 

5.6.5.10.2.2 Requirement per type of claim and test methods 

A laboratory test is not required for product authorisation but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided. 

5.6.5.10.2.2.1 Products intended for use as repellents 

The efficacy of a product should be shown in either a robust simulated-use test, where animals 
can be tethered and observed close by, or a field test where the animals are loose/untethered 
and  observed  from  a  distance  to  establish  the  efficacy  and  persistence  of  the  product  when 
used according to the label in a real-life situation. 

For PT19 products which are applied on animals, no common protocols are available. However, 
the EMA has published several guidelines for the evaluation of ectoparasiticides, see Appendix 
18.  These  guidelines  may  be  adapted  for  the  demonstration  of  the  efficacy  of  repellent 
products to be applied on animals, but any well documented protocol can be used, provided it 
is scientifically sound. 

It is important to give a detailed description of the circumstances in which the experiments are 
conducted, with special emphasis on the ratio of treated to untreated host animals at each trial 
location and trial replicate. Stable flies are forced to get blood meals, and therefore it may be 
of consequence for the trial outcome whether all hosts on a trial location are treated, compared 
to  a  situation  where  only  a  minor  part  of  the  hosts  are  treated  leaving  alternative  untreated 
possibilities for the flies to get blood. It is also of importance that the diurnal rhythm of blood 
sucking activity of stable flies is taken into consideration in the setup of the efficacy trials and 
reported  in  detail.  If  the  product  is  part  of  a  push-pull  strategy,  then  this  setup  must  be 
reported in detail as well. 

If treated horse rugs or similar covers are claimed to be effective in the stable, then it must be 
demonstrated effective in the stable. It is not important to make an evaluation of the flies on 
the  rug  itself,  but  the  emphasis  must  be  on  the  exposed  parts  of  the  host  such  as  the  neck, 
head,  legs,  and  belly  including  the  sheath  on  males  and  udder  on  females,  which  are  thin-
skinned areas and very attractive to several blood-feeding species. 

Tests must be presented with all host animal species claimed. Especially in tests with horses, 
attention is drawn to the fact that horses react very sensitively to biting stable flies and often 
interfere  with  the  counting  as  they  try  to  avoid  the  biting  flies.  It  may  be  necessary  to  make 
more repetitions with horses than with cattle or in other ways adjust the trial setup.  

5.6.5.10.2.2.1.1 Simulated-use test 

In a simulated-use test different parameters are controlled. A simulated-use test of a repellent 
with full control of host animals and absence of their normal avoidance behaviour can be used 
to  evaluate  the  efficacy  and  the  persistency  of  the  treatment;  topically,  spatially  or 
impregnated  textiles.  The  product  should  be  applied  indoors  according  to  the  claim. 
Environmental conditions must be specified at the beginning and during the test (temperature, 

 
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humidity, photoperiod). No specific temperature or humidity are recommended, but the stable 
flies  must  be  present  in  sufficient  numbers,  to  be  able  to  compare  the  treated  and  untreated 
group. A control treatment without repellent should be included in all trials to secure a measure 
of the activity. 

A simulated-use test must be conducted indoor at a time in the season, as well as during the 
day  where  stable  fly  activity  is  sufficient  in  cowsheds,  pigsty,  etc.  Depending  on  the  target 
animal  species,  several  factors  (e.g.  hair  length,  the  thickness  of  the  coat,  grooming,  etc.) 
might  impact  the  efficacy  of  topical  repellents.  Explain  in  the  test  report/documentation  the 
circumstances  around  the  choosing  of  the  test  animals.  If  animals  of  different  colour  are 
chosen be sure not to have a majority of light-coloured animals. Ideally, light coloured animals 
are not included, but state on the label if the product is for a specific colour of animals, if only 
light coloured animals are tested a sound justification is needed to include all fur colours in the 
claim. For products only tested on e.g. pied (black or red) cattle, it can be necessary to state 
on the label that the products are for pied (black or red) cattle. For products tested on sheep it 
can be necessary to test on both sheared and unsheared sheep (a sound justification is needed 
if only sheared or unsheared sheep are tested). 

At least two groups of host animals must be used, one group treated with the product and one 
group  with  no  treatment.  At  least  ten  different  individuals  are  suggested  to  be  used  in  each 
group. In the case of horses, a setup could be 2x5 horses with a treatment switch after e.g. a 
week (depended  on the residual efficacy  of the tested product) tested at one location,  or 2x5 
animals  tested  at  two  different  locations.  At  the  end  of  the  test  period,  there  must  be  2x10 
valid datasets. Each treatment group should be housed in separate enclosures throughout the 
course of the trials to prevent cross infestation or cross-contamination. 

Preferably, no test animals should have a recent history of treatment with insecticide/acaricide 
or  antiparasitic  agents.  Concerning  endo-antiparasitic  agents  this  is  acceptable,  but  details 
(product,  dose,  application  rate,  application  method,  date  of  the  last  treatment,  and  residual 
efficacy) must then be given in the report; the controls and the treated groups must have the 
same status. A safety margin for external applied anthelmintics is the end of last application + 
1 month. The history of the treated and the control groups should be comparable.  

Animals should be treated with the test product once the infestation has become  established. 
The infestation should be documented by e.g. recordings of the numbers of flies, photographs. 
Collections  or  counts must be carried out in the same area  on all hosts, typically  on the legs. 
Inside  barns  and  stables,  it  may  be  difficult  to  distinguish  between  Musca  domestica  and  S. 
calcitrans  at  a  distance  and  it  is  therefore  acceptable  that  counts/collections  are  done  on  a 
tethered  host.  If  the  product  is  acting  at  a  very  close  range  it  may  be  possible  to  make  the 
evaluation  by  treating  one  hind  leg  and  one  front  leg  and  then  compare  the  difference  in  fly-
load  between  the  treated  and  untreated  legs.  The  advantage  of  such  a  setup  is  the 
neutralisation  of  individual  host  differences  in  fly-attraction.  The  attractiveness  of  the  test 
animals or experimental groups should be tested prior to treatment. 

Percent inhibition (landing or feeding) is based on the number of stable flies landing/feeding on 
the  untreated  control  animals  (or  room)  and  the  number  of  stable  flies  landing/feeding  on 
treated animals (room):  

Pct inhibition: ((LandingFeedingcontrol–LandingFeedingtreated)/LandingFeedingcontrol)×100. 

Alternatively  or  in  addition  to  fly  landing  catches,  also  the  frequency  of  certain  horse’s 
avoidance  behaviour  according  to  Mottet  et  al.,  see  Appendix  18,  can  be  recorded  as  a 
substitute, provided the abundance of the target species predominantly causing the avoidance 
behaviour is measured at the beginning and the end of the test. 

5.6.5.10.2.2.1.2 Field trial 

In  a  field  trial  the  animals  must  have  the  opportunity  to  display  normal  avoidance  behaviour 
considering how the product is intended to be used. The tests may be conducted as described 
for  the  simulated-use  test  above,  with  the  adjustment  that  there  is  no  interference  with  the 
host animal’s normal behaviour in the stable. 

The field trials should be conducted on two different geographical locations. 

At least two groups of host animals must be used, one group treated with the product and one 
group  with  no  treatment.  At  least  ten  different  individuals  are  suggested  to  be  used  in  each 

 
 
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group.  Due  to  feasibility  and  cost-effectiveness,  it  would  be  possible  to  use  10  animals  one 
week (5 each in the treatment and control groups) and then use the same individuals again in 
the  second  week,  but  swap  treatments.  Each  animal  would  then  act  as  its  own  control. 
However,  any  residual  activity  of  the  product  has  to  be  excluded.  The  field  trials  can  be 
performed twice with 5 animals (in each of the control and treatment groups) each time at the 
same location, or with 5 animals (in each of the control and treatment groups) at two different 
locations.  At  least  10  animals  (each  as  control  and  treatment)  must  be  tested.  The  two 
locations can be in the same country or region, e.g. two villages. In the case of cattle, the test 
can  be  performed  at  only  one  location  using  10  cows  for  the  control  and  10  cows  for  the 
treatment. 

As  documentation  for  persistency,  counts  are  repeated  with  chosen  intervals  of  lengths 
dependent on the claim. If there is a  claim for use on wet/sweating hosts, then data must be 
provided  showing  that  the  product  is  effective  under  such  circumstances.  The  guideline  from 
the  EMA:  “Guideline  on  specific  efficacy  requirements  for  ectoparasiticides  in  cattle” 
(CVMP/625/2003), and Herholz et al. may be of inspiration for field trial setup, see Appendix 18.  

5.6.5.10.2.2.2 Attractants without PT18 active substances 

Use of an attractant in  a trap in  a push-pull setup may be suggested. A trap could be loaded 
with CO2, heat, octenol, butyric acid, black colour ball, etc. In such a setup the fly load will be 
measured as described above. 

For  product  authorisation  purposes,  the  efficacy  of  an  attractant  in  a  trap  without  an  active 
substance according to PT18 should be proven in a simulated use test or a field test according 
to the instructions for use. 

The  attractants,  e.g.  the  traps,  used  for  the  test  should  be  identical  to  the  product  to  be 
marketed (see Introduction,  section 5.6.5.1.3.7). Traps  should be tested on their  own,  with a 
control tested separately in a similar test setting. 

5.6.5.10.2.2.3 Attractants in combination with PT18 bait products 

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods).  

5.6.5.10.3 Assessment of authorisation 

5.6.5.10.3.1 Norms and criteria 

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for stable flies on livestock in the following way: 

Products intended for use as repellent on animals (including a delivery system) 

A  simulated  use  test  or  a  field  test  demonstrating  ≥80%  repellency  within  the  test  period 
(according  to  the  claim),  directly  after  product  application  and  at  the  end  of  the  claimed  use 
period. 

All claimed target host animals must be tested. 

In  case  of  specific  claims  (e.g.  effective  at  high  temperature,  in  contact  with  water,  host 
sweating, etc.), these should be demonstrated. 

Products  intended  for  use  as  a  space  repellent  or  for  use  as  attractants  without  PT18  active 
substances 

A  trap  with  an  added  attractant  should  catch  significantly  more  stable  flies  than  one  without 
the attractant. The traps used for the test should be identical to the product to be marketed.  

A  simulated  use  test  or  a  field  test  demonstrating  ≥  80  %  efficacy  within  the  test  period 
according  to  the  claim,  directly  after  product  application  and  at  the  end  of  the  claimed  use 
period.  

For  attractants  at  least  a  ratio  of  4:1  of  test  individuals  trapped  in  the  trap  with  attractant 
compared  to  the  control  trap  within  the  test  period  (or  according  to  the  claim),  from  the 
beginning and until the end of the claimed efficacy period.  

All claimed target host species must be tested. 

 
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The  product  label  and  SPC  should  state  that  the  entire  target  insect  population  might  not  be 
caught/repelled  by  this  type  of  product;  the  label  claim  should  be  “reduces”  and  not 
“protects”/“protection”, with no mention of a CPT. 

5.6.5.11 Stored goods-attacking insects and mites 

5.6.5.11.1 Introduction  

The main purpose of biocidal products against stored goods-attacking and infesting insects and 
mites is to: 

• 

• 

• 

control pests in storerooms, freight and alternative transport containers for products of 
plant origin or for other goods; 

protect the actual stored goods against damage or contamination by insects and mites; 

disinfest empty storerooms. 

Products  related  to  product-type  PT19  in  this  case  are  repellents  and  attractants  used  to 
control  harmful  stored-products  pests  by  repelling  or  attracting,  e.g.  mass  trapping,  i.e. 
reducing target organism numbers by luring them in large numbers to a trap that contains an 
attractant and killing the target organisms either with a biocide (attract-and-kill, PT18 product) 
or a devise that bars their exit (PT19 product), or mating disruption. 

The biocidal products are mainly used to achieve hygiene, consumer protection and food-/feed-
/material-  or  stored  goods-protection.  The  term  “stored”  in  this  regard  refers  specifically  to 
stored  products  for  human  use  and  consumption,  and  animal  feed  and  industrial  processing. 
During  storage,  transportation,  processing  and  trade  stored  goods  are  exposed  to  many 
hazards. Biological threats such as insects and mites cause feeding damage and contamination. 
Contamination  may  be  caused  by  living  and  dead  mites,  beetles  and  moths,  their  larvae  and 
eggs  as  well  as  mould  growth  as  a  secondary  infestation.  This  may  lead  to  spoilage  or 
reduction  in  quality  and  imply  a  health  risk  for  humans  and  animals.  An  infestation  is  often 
difficult  to  detect  because  the  organisms  are  very  small,  and  some  develop  inside  the  goods. 
Sometimes  the  infestations  are  only  noticed  by  the  consumer  once  the  insect  leaves  e.g.  the 
food  product  and  enters  the  home  environment.  Therefore,  in  industrial  premises  monitoring 
traps are common to detect an infestation at the initial stage. 

Biocidal products against stored goods-attacking and infesting insects and mites are applied in 
processing facilities and enterprises, along the supply chain and at the consumer site, e.g. uses 
in  storerooms,  warehouses,  food/feed  industry,  private  households,  public  buildings,  cargo 
compartments  on  ships  or  airplanes  and  other  transport  containers  for  processed  products. 
Application  can  take  place  in  empty  as  well  as  in  stocked  rooms  depending  on  the  intended 
use. 

Regarding the control of stored-goods attacking insects and mites it has to be mentioned that 
protection of goods of plant origin is a borderline case: products against stored goods-attacking 
insects  can  be  categorized  as  either  biocides  or  plant  protection  products  depending  of  the 
purpose  of  the  intended  use.  In  general,  uses  controlling  harmful  organisms  in  unprocessed 
plant  products  of  plant  origin  or  those  which  undergo  only  simple  preparation  fall  under  the 
regulations  of  plant  protection,  unless  the  uses  are  considered  to  be  for  reasons  of  hygiene 
rather  than  for  the  protection  of  plants  or  plant  products  (EC/1107/2009).  Aspects  of  plant 
protection  and  plant  protection  products  are  not  covered  by  this  guideline,  but  the  control  of 
insects and mites for reasons of food/feed security, processed goods and materials hygiene is. 

Compared to repellents the use of attractants against stored goods-attacking insects and mites 
in  stored-product  protection  is  already  common,  e.g.  dispensers  in  bakeries  or  pheromone 
moth-traps in households. 

5.6.5.11.1.1 Target and test organisms 

Among  the  organisms  attacking  stored  goods  certain  groups  of  organisms  are  of  particular 
importance:  beetles  (especially  weevils),  moths,  dust-lices  and  mites.  Some  of  the  most 
common  pests  that  infest  stored  plant  products  on  their  way  from  primary  production  via 
processing industry, transportation and trade to the consumer are listed and suggested as test 
organisms  in  EPPO  guidance.  EPPO  focuses  on  insects  and  mites  known  as  typical  harmful 
organisms of stored-plant products. However, these arthropods could also infest processed and 
packaged products like cereal mixtures, dried fruits and vegetables, processed cereal products, 

 
 
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bakery products, chocolate, confectionary and pasta, expeller, nuts, pulses, oilseeds, herbs, tea 
etc.  Some  of  them,  e.g.  Dermestidae  (especially  their  larvae)  could  also  live  or  exist  in  dried 
products of animal origin like milk powder and meat. 

This is a non-exhaustive list of relevant target organisms: 

Beetles (including weevils) such as: 

Acanthoscelides obtectus – Bean weevil 

Alphitobius diaperinus – Lesser mealworm / litter beetle 

Anobium punctatum – Common furniture beetle 

Anthrenus flavipes – Furniture carpet beetle 

Anthrenus munroi 

Anthrenus museorum – Museum beetle 

Anthrenus pimpinellae – Bird nest carpet beetle 

Anthrenus scrophulariae – Buffalo carpet beetle 

Anthrenus verbasci – Carpet beetle 

Attagenus unicolor – Black carpet beetle 

Callosobruchus maculatus – Cowpea weevil 

Cryptolestes ferrugineus – Rusty grain beetle 

Cryptolests busillus – Flat grain beetle 

Dermestes lardarius – Larder beetle 

Hylotrupes bajulus – House longhorn beetle 

Lasioderma serricorne – Tobacco beetle 

Lyctus brunneus – Brown powderpost beetle 

Oryzaephilus surinamensis – Sawtoothed grain beetle 

Rhyzopertha dominica – Lesser grain borer 

Sitophilus granarius – Wheat weevil 

Sitophilus oryzae – Rice weevil 

Sitophilus zeamais – Maize weevil 

Stegobium paniceum – Drugstore beetle 

Tenebrio molitor – Yellow mealworm 

Tenebroides mauritanicus - Cadelle 

Tribolium castaneum – Red flour beetle 

Tribolium confusum – Confused flour beetle 

Trogoderma granarium – Khapra beetle 

Trogoderma inclusum – Larger cabinet beetle 

Trogoderma longisetosum 

Trogoderma variabile 

Moths such as: 

Cadra cautella (Ephestia cautella) – Almond moth / tropical warehouse moth 

Corcyra cephalonica – Rice moth 

Endrosis sarcitella – White-shouldered house moth 

Ephestia elutella – Cacao moth/Tobacco moth 

Ephestia kuehniella – Mediterranean flour moth 

 
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Hofmannophila pseudospretella – Brown house moth 

Nemapogon granella – European grain moth 

Plodia interpunctella – Indian meal moth 

Pyralis farinalis – Meal moth 

Sitotroga cerealella – Angoumois grain moth 

Booklice/dustlice such as: 

Liposcelis corroden 

Liposcelis decolor 

Dorypteryx domestica 

Mites such as: 

Acarus siro – Flour mite 

Lepidoglyphus destructor – Storage mite 

Tyrophagus longior – Mould mite 

Tyrophagus putrescentiae – Cheese mite 

5.6.5.11.2 Dossier requirements  

General dossier requirements are stated in the General Introduction (see section 5.6.5.1.3). 

5.6.5.11.2.1 Test species and conditions 

The (groups of) organisms included in the group of stored goods-attacking insects and mites do 
not  belong  to  a  single  monophyletic  group,  but  to  different  classes  (insects  and  mites 
(arachnida)) or orders, e.g. beetles and booklice. Therefore, general claims at such a high level 
are difficult to substantiate. In practice, products for use against stored goods-attacking insects 
and mites will typically cover the control of pests for a specific type of stored goods, e.g. flour, 
grain, tobacco, processed cereals, paper, leather. 

Due  to  the  diverse  nature  (biology,  ecology,  habitat,  diet,  behaviour,  etc.)  of  the  organisms 
included in the group of stored-goods attacking insects and mites, and due to the specificity of 
active  substances  such  as  pheromones,  efficacy  can  only  be  claimed  against  the  species  for 
which  efficacy  was  demonstrated  in  efficacy  studies.  Nevertheless,  general  claims  for  smaller 
groups  which  consist  of  specific  organisms  and  a  specific  type  of  stored  good  to  be  protected 
could  be  authorised.  This  is  the  case,  for  example,  when  different  moth  species  may  produce 
the  same  pheromone  components  for  the  attraction  of  sex  mates.  Semiochemicals  are  often 
pest  specific  and  act  by  modifying  behaviour.  In  the  case  semiochemicals  that  have  multiple 
targets,  extrapolation  to  a  group  of  related  species  is  possible.  A  schematic  representation  of 
the  extrapolation  possibilities  on  effectiveness  is  available  in  the  EPPO  standard  PP1/296. 
Alternative  extrapolations  may  be  proposed  by  the  applicant.  A  clear  justification  is  always 
necessary and may be supported by scientific literature and/or data. For semiochemicals EPPO 
Standard PP1/264 has specific advice on mating disruption pheromones, including a schematic 
representation  of  extrapolation  possibilities  given  in  PP1/296.  For  extrapolation  from  efficacy 
data  for  one  species  to  a  limited  group  a  robust  justification  based  on  scientific  literature 
and/or data should be provided. 

 
 
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Table 28: Examples of different pheromones that are used to attract food moths. 

Group 
organisms 

of 

target 

Pheromone 

Species  against  which  efficacy  should  be 
demonstrated 

(Z, 
E)‐9,12‐tetradecadien‐1‐yl 
acetate (TDA) 

Ephestia kuehniella 

Ephestia elutella 

Plodia interpunctella 

(Z, 
E)‐7,11‐hexadecadien‐1‐yl 
acetate (HDA) 

Sitotroga cerealella 

Food moths or 
storage moth species 
(e.g. processed 
cereals, dried fruits, 
flour, processed 
nuts, infusions) – 
Pyralidae  

Food moths or 
storage moth species 
(e.g. processed 
cereals, dried fruits, 
flour, processed 
nuts, infusions) – 
Gelechiidae 

For  general  claims,  apart  from  efficacy  studies,  a  justification  based  on  robust  scientific 
information,  e.g.  scientific  literature,  has  to  be  provided,  demonstrating  efficacy  against  the 
organisms covered by the claim, e.g. by demonstrating all moth species covered by the claim 
use the same pheromone. 

General  claims  should  include  both  the  group  of  organisms,  e.g.  a  specific  genus  of  beetles, 
moths, to be controlled and the type of stored goods to be protected. If multiple general claims 
are  made,  efficacy  should  be  demonstrated  for  each  of  these  claims.  Examples  of  possible 
general claims and the species against which efficacy should be demonstrated are presented in 
the tables below.  

The mentioned target groups, species and goods to be tested or protected are only examples. 
It  is  advised  to  discuss  with  the  competent  authority  the  acceptability  of  any  general  product 
claims,  with  regard  to  target  organisms  or  goods  to  be  protected,  prior  to  product  dossier 
submission. 

Table 29: General claims 

General claims based on target organisms 

Group 

Species against which efficacy should be demonstrated 

Flour moths 

Ephestia cautella 

Plodia interpunctella or Ephestia kuehniella 

Grain (products) beetles  Cryptolestes busillus or Cryptolestes ferrugineus 

Oryzaephilus surinamensis 

Tenebroides mauritanicus or Tenebrio molitor  

Grain weevils 

Sitophilus granarius  

Sitophilus zeamais or Sitophilus oryzae 

 
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General claims based on goods to be protected 

Good to be protected 

Species against which efficacy should be demonstrated 

Stored grain 

Sitophilus granarius 

Rhyzopertha dominica  

Oryzaephilus surinamensis  

Cryptolests busillus or Cryptolestes ferrugineus 

Sitophilus granarius  

Sitophilus zeamais or Sitophilus oryzae 

Tenebroides mauritanicus or Tenebrio molitor 

Tribolium sp. 

Plodia interpunctella or Ephestia kuehniella 

Sitotroga cerealella 

Flour and flour products  Tribolium sp. 

Oryzaephilus surinamensis  

Sitophilus zeamais or Sitophilus oryzae  

Tenebroides mauritanicus or Tenebrio molitor 

Plodia interpunctella or Ephestia kuehniella 

Trogoderma granarium 

Lasioderma serricorne 

Stegobium paniceum  

Ephestia kuehniella 

Spices and Tobacco 

Lasioderma serricorne 

Stegobium paniceum  

Dermestes sp. 

Dehydrated foods 

Plodia interpunctella  

Dermestes sp. 

Trogoderma granarium 

Nuts and dried fruit 

Plodia interpunctella or Ephestia cautella 

Oryzaephilus surinamensis 

Tribolium sp. 

Dried peas and beans 

Acanthoscelides obtectus 

Callosobruchus maculatus 

The  applicant  has  to  describe  the  target  organisms  and  any  special  details  depending  on  the 
species and relevant for the efficacy of the biocide, e.g.: 

•  Sex and status of used insects/mites; 

•  Nature of communication; 

•  Developmental stages (eggs, larvae, pupae, diapausing larvae, imagines); 

•  Mating/calling behaviour and season, polygamous males; 

 
 
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•  Special  characteristics  like  flying  in  dawn  light,  living/developing  inside  the  cereal,  egg 

laying sites, seasonal aspects, movement distances of adults; 

•  Resistance  risk  and  appropriate  management  strategies,  if  relevant  (see  also  EPPO 

guidance). 

With regard to test conditions, the following aspects, if relevant, should be considered: 

•  Test room/space dimensions should be representative of intended use; 

• 

Information  on  the  used  methods  should  be  provided  (e.g.  mass-trapping,  mating 
disruption, “attract and kill”); 

• 

Is the intended use to attract or repel one sex or both; 

•  Devices (e.g. spraying cans / traps) used to apply the product in efficacy tests should be 
the same as for the product to be  authorised (specify design, color and position in test 
setup); 

•  Prevent interference between treatment rooms/plots; 

•  Treatment with different dosages and pest densities, if relevant;  

•  Climate  data  should  be  recorded:  e.g.  air  dynamics  in  the  test  facility,  temperature 
(range  of  change  and  or  monthly  mean),  humidity  (range  of  change  and/or  monthly 
mean). Climatic conditions should be in accordance with the intended area of use. 

For pheromone attractants: 

•  There  may  not  always  be  a  good  correlation  between  trap  catches  and  subsequent 
infestation/crop damage when using the same sex pheromone for monitoring traps and 
mating disruption:  

-  males could not locate traps in a pheromone area, or;  

- 

they could be back-caught while the dispensed pheromone level is too low; 

-  deflect attention from females; 

-  alternative approach: traps with other semiochemicals (kairomones) for the trap 

lures, light traps or water traps (for Ephestia moths); 

•  Development  of  the  population  of  the  pest  organism  (depends  on  reproduction, 

nutrition, environmental conditions) should be monitored; 

•  Avoid  immigration  of  mated  females  to  avoid  test  disturbance  by  reproduction.  For 

simulated-use test virgin females should be used, if relevant.  

Please  note  that  the  parameters  above  are  examples,  the  relevance  of  each  of  these  is 
dependent on the product to be authorised and claim(s). 

5.6.5.11.2.2 Requirements per type of claim and test methods 

In the product claim, it should be specified whether the product is intended to treat an ongoing 
infestation or to prevent new infestations. Depending on the intended use, e.g. the application 
and  the  purpose  of  the  product  testing  requirements  are  listed  in  sections  5.6.5.11.2.2.1  and 
5.6.5.11.2.2.2. For both simulated-use tests and field studies, a minimum of 5 valid replicates 
(different  test  sites/locations/buildings  in  the  case  of  a  field  study)  is  required.  For  each 
claimed  condition  of  use  (indoor,  outdoor,  cupboard,  storage  room)  5  replicates  should  be 
performed.  

A  laboratory  test  is  not  required  for  product  authorisation  but  can  be  used  to  evaluate  if  the 
right  dose  is  chosen  and  to  see  whether  the  product  repels  under  ideal  conditions.  For  active 
substance  approval  only,  a  laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be 
waived for active substance approval when a suitable simulated-use test is provided. 

For simulated-use tests with sex pheromones, both sexes should be released as in nature the 
traps  will  compete  with  the  pheromone  released  by  the  females.  A  food  source  should  be 
present  when  measuring  reproduction  (F1  generation)  and/or  when  reinfestation  of  stored 
goods is relevant for the label claim. 

 
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For  simulated-use  tests  with  alimentary  attractants,  an  alternative  food  source  should  be 
offered in the test setup. The time needed to reach the claimed efficacy has to be indicated. 

Due  to  the  diverse  nature  (biology,  ecology,  habitat,  diet,  behaviour,  etc.)  of  the  organisms 
included in the group of stored-goods attacking insects and mites, and due to the specificity of 
active  substances  such  as  pheromones,  efficacy  can  only  be  claimed  against  the  species  for 
which efficacy was demonstrated in efficacy studies.  Only developmental stages against which 
efficacy was demonstrated can be authorised. For a general claim additional data needs to be 
provided, e.g. from the scientific literature, demonstrating the acceptability of the claim. 

In  the  simulated-use  tests,  the  number  of  test  organisms  should  be  proportional  to  the  room 
size (relevant pest density) and sufficient to draw statistically robust conclusions. 

5.6.5.11.2.2.1 Products for consumer (non-professional) use 

For consumer products, simulated-use tests are required. Simulated-use tests can be waived if 
a robust field trial is submitted. A simulated-use test can be a test, performed in a laboratory, 
where  insects  or  mites  (either  cultured  or  natural  populations)  are  in  contact  with  the  stored 
goods, e.g. breakfast cereal, flour) and the biocide is applied according to the instructions for 
use.  

A  control  treatment  in  parallel  without  biocide  with  the  same  number  of  replicates  should  be 
included in all simulated-use trials. Tests should be performed in space/room of a size relevant 
for the intended use.  

5.6.5.11.2.2.2 Products for professional use 

For  products  for  professional  use  field  tests  are  required.  Given  the  sometimes  large  and 
complex  environments,  e.g.  warehouses,  where  products  are  to  be  used,  field  tests,  e.g.  on 
existing infestations or with known numbers of test organisms and stages are mandatory when 
it comes to demonstrating efficacy, especially in the case of mating disruption.  For field tests, 
trials  should be conducted under  representative  conditions,  e.g.  food  moth in  food  factory, or 
insect  pest  of  animal  or  vegetable  product  in  the  storeroom,  etc.  In  case  residual  efficacy  is 
claimed, efficacy data should be submitted to support the claimed residual efficacy period after 
product application (opening of the product). 

5.6.5.11.2.2.3 Attractants in PT18 bait products 

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for  bait 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods). 

5.6.5.11.3 Assessment of authorisation  

5.6.5.11.3.1 Norms and criteria  

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. Indicators of effectiveness for stored goods-attacking insects and mites are, 
e.g.: 

•  Population reduction; 

•  Control of population target (e.g. sex, developmental stage): male or female catches in 
relation to the released vital population; behaviour of untrapped /remaining males (e.g. 
impaired in the finding capability); 

•  Damage of products, population reservoir (if known). 

Moderate  effectiveness  may  not  be  acceptable  because  of  the  risk  of  consolidating  the 
infestation and the carry-over with traded goods. 

This is implemented for stored goods-attacking insects and mites in the following way: 

Attractants 

•  ≥80%  population  target,  e.g.  sex,  developmental  stage,  attraction  compared  to  the 
negative  control  for  laboratory,  simulated-use  and  field  trials.  Efficacy  should  be 
demonstrated within the test period (or according to the claim), from the beginning and 
until the end of the claimed efficacy period; 

 
 
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•  at least a ratio of 4:1 of test individuals trapped in the trap with attractant compared to 
the control trap within the test period (or according to the claim), from the beginning 
and until the end of the claimed efficacy period. 

For  products  intended  for  use  as  attractant  in  combination  with  PT18  (bait  products),  the 
efficacy  evaluation  should  be  done  on  the  basis  of  the  requirements  for  products  (see  PT18 
Chapter: Insecticides, acaricides and products to control other arthropods). 

Repellents 

•  ≥80% repellency (influence on population dynamics, population reduction, influence on 
population  target)  for  laboratory,  simulated-use  and  field  trials.  Efficacy  should  be 
demonstrated  within  the  test  period  (or  according  to  the  claim),  at  the  beginning  and 
until the end of the claimed efficacy period. 

Proof  of  non-insecticidal  efficacy  has  to  be  examined  in  a  simulated-use  test  if  it  cannot  be 
waived (for details see Introduction, section 5.6.5.1.3.8). After the trial insects are maintained 
in the test system to observe whether insecticidal effects could be caused by contact/exposure 
to the test repellent product. Mortality of the insects/mites should be monitored after 24 hours.  

Deviations from these norms are possible but should be justified in the application, e.g. data on 
protection of the stored goods may be used if 100% protection was demonstrated. 

5.6.5.12 Textile-attacking insects (including fur and fabric attacking insects) 

5.6.5.12.1 Introduction  

The purpose of biocidal repellents  or attractants against textile-attacking insects is to prevent 
or  to  combat  pest  infestation  of  textile  articles  especially  those  manufactured  from  natural 
fibres like wool, hair, feathers or fur and also blends of natural and synthetic fibres. 

Repellents  against  textile-attacking  insects  can  also  be  incorporated  in  the  textile  by  industry 
for preventive treatments.  

5.6.5.12.1.1 Biology  

The  two  main  orders  containing  textile-attacking  insect  species  are  Lepidoptera  (moths)  and 
Coleoptera  (beetles).  The  webbing  clothes  moth  (Tineola  bisselliella),  case-making  clothes 
moth  (Tinea  pellionella),  brown  house  moth  (Hofmannophila  pseudospretella)  and  carpet 
beetles  (Anthrenus  spp.,  Anthrenocerus  spp.,  Attagenus  spp.,  Trogoderma  spp.)  are  common 
in-house pests that feed on clothing, drapery, carpet and other natural hair fibres. The larvae 
of these insects feed on natural hair fibres, which provide protein from keratin in the hair. They 
have adapted to be able to digest keratin, which is not easily digested by other insects. 

Clothes moths are distributed worldwide. They feed during the larval cycle either within a silken 
cocoon  attached  to  hair  fibre  (only  T.  bisselliella)  or  inside  a  portable  silken  case  (all  other 
textile moth species). Clothes moths larvae that feed only on natural hair fibres such as wool, 
will not feed on pure silk, pure cotton, pure linens or pure synthetic fibres. Adult clothes moths 
do not feed. After mating the females lay eggs directly on the natural fibre food source.  

Carpet  beetle  larvae,  e.g.  Anthrenus  sp.,  Anthrenocerus  sp.,  Attagenus  sp.  attack  woollens, 
rugs  and  upholstered  furniture,  among  other  things.  The  adult  beetles,  which  feed  on  nectar 
and  pollen,  can  enter  the  home  on  plants,  flowers  or  other  vegetation.  Bird  nests  and  animal 
burrows  which  may  be  found  in  or  close  to  houses  may  also  provide  an  entry  path.  In  the 
context  of  museums  there  is  likely  an  entrance  through  artefacts  brought  in  from  other 
museums  with  an  insect  pest  problem.  Eggs  are  then  laid  on  lint  in  protected  areas  such  as 
behind  baseboards.  Once  hatched,  larvae  begin  feeding  on  a  number  of  natural  textiles  or 
displays (animal horns, hoofs, insect collections, etc.). 

5.6.5.12.2 Dossier requirements  

Dossier requirements are stated in the Introduction (see section 5.6.5.1.3). 

5.6.5.12.2.1 Test species  

A product against textile-attacking insects should be tested on: 

At least one of the following moth species: 

• 

the webbing clothes moth (Tineola bisselliella) 

 
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• 

• 

the case-making clothes moth (Tinea pellionella) 

the brown house moth (Hofmannophila pseudospretella) 

At least one of the following carpet beetle species: 

• 

• 

• 

Anthrenus spp. 

Attagenus spp. 

Anthrenocerus spp. 

For a general claim “against adult textile-attacking insects” adult target organisms, moths and 
carpet beetles, should be tested. For general claims “against adult textile-attacking moths” or 
“against adult textile-attacking carpet beetles” adult moths or carpet beetles should be tested, 
respectively. If products are exclusively claimed against larvae of moths and/or carpet beetles, 
then  the  efficacy  should  be  demonstrated  for  that  developmental  stage  (instead  of  adults). 
Deviations should be justified and tested. 

Due to the specificity of certain active substances, e.g. pheromones, for products based on an 
active  substance  with  a  species-specific  mode  of  action,  only  effects  against  textile-attacking 
insect species that have been tested under simulated-use and/or field conditions, depending on 
the type of claim, should be claimed on the product label and in the SPC. 

The  composition  of  the  material  to  be  protected  should  be  indicated  on  the  label,  to  avoid 
application of products on materials which are not attractive to textile-attacking moths and/or 
carpet beetles. 

5.6.5.12.2.2 Requirements per type of claim and test methods 

A laboratory test is not required for product authorisation but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided.  

5.6.5.12.2.2.1 Repellent products against textile attacking insects  

In  most  cases,  these  products  will  not  be  able  to  eliminate  an  existing  infestation.  They  are 
only useful as a preventive measure. Unless otherwise proven in efficacy trials, the label should 
include  wording  such  as:  “Ensure  there  is  no  infestation  in  place  before  the  use  of  this 
repellent. Repellents should only be used as a preventive measure”. 

For  product  authorisation  purposes  the  efficacy  of  repellent  products  should  be  proven  in  a 
simulated-use  test  according  to  the  instruction  for  use  (test  design  examples  see 
5.6.5.12.2.2.1.3 and 5.6.5.12.2.2.1.4). 

The repellents used for the test should be identical to the product to be marketed. 

Proposed label claims regarding the performance of the product must be simulated in the study 
(for details see Introduction, section 5.6.5.1.3.5). 

For products intended to be used as  space treatment  the room size stated in the SPC  and on 
the  label  must  be  used  in  the  efficacy  trial.  For  the  extrapolation  to  larger  room  sizes  than 
proven in the efficacy test a justification is necessary. 

5.6.5.12.2.2.1.1 Laboratory choice test for repellents 

The  repellence  of  an  active  substance  can  be  tested  in  a  choice  test,  examples  see  Appendix 
18: Beerwinkle K.R., et al. and Arnault I., et al.. Insects are placed in a tunnel or a release box 
interconnected with tunnels between two dark boxes, both containing fabric made of a material 
which  is  in  accordance  with  the  claim.  One  of  the  boxes  contains  the  repellent  product.  The 
application quantity of the product must match the size of the boxes. The insects (at least 10) 
are released in the tunnel or release box, then they can choose the treated or control box. The 
ratio of insects found in the treated vs. control box is a measure of the efficacy of the product. 
Insects in the tunnel that have not chosen a side (control or treated box) should not be used in 
the  evaluation  of  the  product.  Saturation  of  the  system  with  repellent  should  be  avoided.  If 
necessary, the system should be ventilated, e.g. air has to be sucked out at the insect’s release 
site and introduced both at the treated and control box. If a product is claimed against one sex 

 
 
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an  equal  number  of  both  sexes  can  be  used  in  the  test,  but  only  the  target  sex  should  be 
evaluated. 

Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative  control) 
should be performed. 

Environmental  conditions  must  be  specified  (and  justified)  for  the  test  itself,  and  during  the 
storage  of  the  treated  substrates/surfaces  or  repellent  product  (temperature,  humidity, 
photoperiod, ventilation) and shall be in accordance with the claim. Please note, the darker the 
test conditions, the better the insects behave and the more realistic the experiments are. 

5.6.5.12.2.2.1.2 Laboratory choice test for oviposition repellent products  

A  choice  test  similar  to  the  test  described  in  section  5.6.5.12.2.2.1.1  can  be  used  with  the 
following adaptations: 

The dark boxes contain fabric attractive for insect  oviposition. The nature  of the  fabric  should 
be  in  accordance  with  the  claim  and  the  fabric  should  be  attractive  to  the  textile-attacking 
insects for oviposition. The attractivity of the fabric must be documented. 

To record the development of potentially laid eggs during the test period the fabrics are placed 
in closed trays and incubated. Breeding conditions must be specified  and justified for the test 
itself and shall be in accordance with the claim. Please note, the darker the test conditions, the 
better the insects behave and the more realistic the experiments are. The degree of repellence 
is shown as the number of eggs on the treated fabric versus the number of eggs on the control 
fabric or alternatively, the number of offspring (larvae) hatched from the treated fabric versus 
the offspring hatched from the control fabric. 

Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative  control) 
should be performed. 

5.6.5.12.2.2.1.3 Simulated-use test for repellents 

Mandatory requirements: 

•  The simulated-use test should reflect the real use situation.  

•  Simulated-use  tests  should  be  conducted  in  closets.  If  the  product  is  claimed  for  an 
application  in  closets  a  minimum  volume  of  0.5  m³  should  be  used,  which  may  be 
divided  into  smaller  compartments.  For  products  to  be  used  in  smaller  compartments, 
like drawers or boxes, tests should be done in containers with a volume according to the 
claim appearing on the SPC.  

•  The  treatment  and  control  group  tests  are  conducted  each  in  a  separate  room.  The 
control serves to show that the test insects are vital and able to infest the closet under 
test conditions.  

•  The degree of repellence is shown by the number of test insects (including the potential 
offspring)  in  the  test  closet  versus  the  number  of  test  insects  (including  the  potential 
offspring) in the negative control closet. 

•  Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative 
control)  should  be  performed.  Each  replicate  consists  of  at  least  50  mature  clothes 
moths or carpet beetles.  

•  Environmental conditions must be specified (and justified) for the test itself, and during 
the  storage  of  the  treated  substrates/surfaces  or  repellent  product  (temperature, 
humidity,  photoperiod,  ventilation)  and  shall  be  in  accordance  with  the  claim.  Please 
note, the darker the test conditions, the better the insects behave and the more realistic 
the experiments are. 

•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8). 

•  For the evaluation of the claim “protects clothing” damage to the test material must be 
documented by photography. Compared to the control setup no damage to the textiles 
of the treated cabinet should be documented. 

•  For residual efficacy, textile-attacking insects are exposed to the product at several time 

intervals after application (including the end of the claimed period). 

 
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Other  test  designs  than  the  following  example  (see  Appendix  18:  Plarre  R.,  et  al.)  can  be 
accepted if the protocol is scientifically valid. Deviations  from this test design,  e.g. number of 
test insects in a lower or higher volume in adoption to the intended uses are possible and must 
be explained. 

Test design:  

One  closet  is  located  centrally  in  a  ventilated  test  room  (Peet  Grady  chamber  or  equivalent 
ventilated  room  with  a  size  of  at  least  1.80  x  1.80  x  1.80  m).  To  mimic  the  practical  use 
situation pieces  of fabric that are attractive to  insects and  which nature is in accordance with 
the  claim  should  be  placed  inside  the  closet.  Furthermore,  the  door  of  the  closet  should  be 
opened with a frequency resembling the normal opening of a closet, once a day for at least 10 
seconds,  to  show  that  this  does  not  reduce  efficacy  during  the  completion  of  the  assay.  The 
closet should provide openings, e.g. a number of holes of 2-4 mm diameter or a longer crevice 
with 2-4 mm width, for entry of the moths or beetles when the door of the closet is closed. 

Sample  trays  filled  with  pieces  of  biocide-free  attractive  fabrics  in  accordance  with  the  claim 
are  deposited  without  a  cover  on  2-3  different  compartments  of  the  closet  each  with  at  least 
one tray. These open test trays are used to monitor egg deposition and offspring development 
of  the  target  organisms.  Products  should  be  applied  according  to  the  claim  (dose,  application 
method). The attractivity  of the fabric must be documented.  No food  source  should be added 
with  the  biocide  free  samples.  This  could  bias  the  results,  or  if  feeding  is  necessary  then 
laboratory food could be provided in both the treated and control samples. 

At  least  50  adult  mature  clothes  moths  or  carpet  beetles  are  released  approximately  1 m  in 
front  of  the  closet  door  into  the  test  room  and  monitored  at  regularly  defined  time  intervals 
until  the  end  of  the  claimed  efficacy  period  (residual  efficacy).  At  each  time  interval,  the  test 
insects should be monitored for several days after releasing into the room, e.g. 5 days after the 
introduction of the product. Test insects can move freely within the test room. Access into the 
closet should be possible. 

At the end of the test-run, insects inside and outside the closets are collected separately.  If a 
product is claimed against one sex an equal number of both sexes can be used in the test, but 
only the target sex should be evaluated.  

The  chamber  and  the  closet  must  be  cleaned  and  dried  between  each  replicate  to  avoid 
chemical contamination and saturation of the product. 

Proof of non-insecticidal efficacy: After product application test insects should be placed in the 
test  closet (or smaller  compartments)  for 1 hour to  observe whether insecticidal effects could 
be  caused  by  contact/exposure  to  the  test  repellent  product.  To  avoid  a  no-choice  test  which 
forces  the  insects  into  contact  with  the  product,  the  insects  should  be  able  to  avoid  direct 
contact  with  the  product.  After  each  test  run,  the  mortality  of  the  adult  insects  should  be 
monitored. 

Efficacy assessment: To record the development of potentially laid eggs during the test period 
the  sample  trays  are  covered  and  incubated.  Breeding  shall  be  conducted  until  the 
development of eggs to adults is completed in the control group. 

The rate of efficacy of a product depends on its capability of preventing textile-attacking insects 
from entering the closet. The repellent effect (%) is calculated by comparing with the negative 
control (insects inside the control closet, number of offspring). 

5.6.5.12.2.2.1.4 Simulated-use test for oviposition repellent products 

A simulated-use test similar to the test described in section (5.6.5.12.2.2.1.3) can be used with 
the following adaptations: 

At the end of the test run only the development of potentially laid eggs during the test period 
must  be  recorded.  The  number  of  insects  inside  and  outside  the  closets  must  not  be 
determined.  The  degree  of  repellence  is  shown  as  the  number  of  eggs  on  the  treated  fabric 
versus  the  number  of  eggs  on  the  control  fabric  or  alternatively  the  number  of  offspring 
(larvae) hatched in the test tray versus the offspring hatched in the control tray. 

 
 
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5.6.5.12.2.2.2 Attractants without PT18 active substances  

For product authorisation purposes the efficacy of attractant products intended for use in traps 
should be proven in a simulated-use test according to the direction of use (test design example 
see 5.6.5.12.2.2.2.2). 

The attractants, e.g. the traps, used for the test should be as similar as possible to the product 
to be marketed (for details see Introduction, section 5.6.5.1.3.7).  

5.6.5.12.2.2.2.1 Laboratory choice test for attractants 

Attractiveness  of  an  active  substance  can  be  tested  in  a  choice  test,  examples  see  Appendix 
18: Beerwinkle K.R., et al. and Arnault I., et al. Insects are placed in a tunnel or a release box 
interconnected with tunnels between two dark boxes, both containing fabric made of a material 
which  is  in  accordance  with  the  claim.  One  of  the  boxes  contains  the  attractant  product.  The 
application quantity of the product must match the size of the boxes. The insects (at least 10) 
are released in the tunnel or release box, then they can choose the treated or control box. The 
ratio of insects found in the treated vs. control box is a measure for the efficacy of the product. 
Insects in the tunnel that have not chosen a side (control or treated box) should not be used in 
the  evaluation  of  the  product.  Saturation  of  the  system  with  attractant  should  be  avoided.  If 
necessary, the system should be ventilated, e.g. air been sucked out at the insect’s release site 
and introduced both at the treated and control box. If a product is claimed against one sex an 
equal  number  of  both  sexes  can  be  used  in  the  test,  but  only  the  target  sex  should  be 
evaluated. 

Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative  control) 
should be performed. 

Environmental  conditions  must  be  specified  (and  justified)  for  the  test  itself,  and  during  the 
storage  of  the  treated  substrates/surfaces  or  repellent  product  (temperature,  humidity, 
photoperiod, ventilation) and shall be in accordance with the claim. Please note, the darker the 
test conditions, the better the insects behave and the more realistic the experiments are. 

5.6.5.12.2.2.2.2 Simulated-use test for attractants 

Mandatory requirements: 

•  The simulated-use test should reflect the real use situation.  

•  Simulated-use  tests  should  be  conducted  in  closets.  If  the  product  is  claimed  for  an 
application  in  closets  a  minimum  volume  of  0.5  m³  should  be  used,  which  may  be 
divided  into  smaller  compartments.  For  products  to  be  used  in  smaller  compartments, 
like drawers or boxes, tests should be done in containers with a volume according to the 
claim appearing on the SPC.  

•  The  treatment  and  control  group  tests  are  conducted  each  in  a  separate  room.  The 
control serves to show that the test insects are vital and able to infest the closet under 
test conditions.  

•  Attraction  depends  on  the  mode  of  action  of  pheromones  by  the  attraction  of  adult 
individuals of a specific sex or by the attraction of males and females (sex-unspecific). 
Therefore, the degree  of attractants could be shown for sex-unspecific products  as the 
trapped number of test insects in a closet containing the trap with the attractant versus 
the  trapped  number  of  test  insects  in  a  control  closet  containing  the  identical  trap 
without the active substance. For products which affect only individuals of one sex, the 
efficacy should be shown on the next generation, i.e. by counting eggs, larvae or pupae 
on fabrics in the treated and control closet. 

•  Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative 
control)  should  be  performed.  Each  replicate  consists  of  at  least  50  mature  clothes 
moths or carpet beetles.  

•  Environmental conditions must be specified (and justified) for the test itself, and during 
the  storage  of  the  treated  substrates/surfaces  or  repellent  product  (temperature, 
humidity,  photoperiod,  ventilation)  and  shall  be  in  accordance  with  the  claim.  Please 
note, the darker the test conditions, the better the insects behave and the more realistic 
the experiments are. 

 
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•  Non-insecticidal  efficacy  must  be  proven,  if  it  cannot  be  waived  (for  details  see 

Introduction, section 5.6.5.1.3.8). 

•  For the evaluation of the claim “protects clothing” damage to the test material must be 
documented by photography. Compared to the control setup no damage to the textiles 
of the treated cabinet should be documented. 

•  For residual efficacy, textile-attacking insects are exposed to the product at several time 

intervals after application (including the end of the claimed period). 

Other  test  designs  than  the  following  example  (see  Appendix  18:  Plarre  R.,  et  al.)  can  be 
accepted if the protocol is scientifically valid. Deviations  from this test design,  e.g. number of 
test insects in a lower or higher volume in adoption to the intended uses are possible and must 
be explained. 

Test  design:  One  closet  is  located  centrally  in  a  ventilated  test  room  (Peet  Grady  chamber  or 
equivalent  room  with  a  size  of  at  least  1.80  x  1.80  x  1.80  m).  To  mimic  the  practical  use 
situation pieces  of fabric that are attractive to  insects and which nature is in accordance with 
the  claim  should  be  placed  inside  the  closet.  Furthermore,  the  door  of  the  closet  should  be 
opened with a frequency resembling the normal opening of a closet (once a day for at least 10 
seconds),  to  show  that  this  does  not  reduce  efficacy  during  the  completion  of  the  assay.  The 
closet should provide openings, e.g. a number of holes of 2-4 mm diameter or a longer crevice 
with 2-4 mm width) for entry of the moths or beetles when the door of the closet is closed. 

Sample  trays  filled  with  pieces  of  biocide-free  attractive  fabrics  in  accordance  with  the  claim 
are  deposited  without  a  cover  on  2-3  different  compartments  of  the  closet  each  with  at  least 
one  tray.  Products  should  be  applied  according  to  the  submitted  claim  on  the  SPC  (dose, 
application method). The attractivity of the fabric must be documented. No food source should 
be added with the biocide free  samples. This  could bias the results,  or if  feeding is necessary 
then laboratory food could be provided in both the treated and control samples. 

At  least  50  adult  clothes  moths  or  adult  carpet  beetles  are  released  into  the  closet.  After  an 
acclimatization period pieces of fabric are replaced, and the trap is applied in accordance with 
the claim appearing on the SPC. 

Number of trapped insects is regularly monitored at defined time intervals until the end of the 
claimed  efficacy  period  (residual  efficacy).  At  each  time  interval,  the  test  insects  should  be 
monitored for several days after releasing into the room, e.g. 5 days  after the introduction of 
the product. A mixed population can be released, but just the target sex should be evaluated. 

The  chamber  and  the  closet  must  be  cleaned  and  dried  between  each  replicate  to  avoid 
chemical contamination and saturation of the product. 

Efficacy  assessment:  The  rate  of  efficacy  of  a  product  depends  on  its  capability  to  attract 
textile-attacking  insects.  The  attraction  (%)  is  calculated  by  comparing  with  the  negative 
control (by counting the number of adult insects in the trap or by the number of offspring).  

5.6.5.12.2.2.3 Attractants in PT18 bait products 

Efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for  bait 
products (see PT18 Chapter: Insecticides, acaricides and products to control other arthropods). 

5.6.5.12.3 Assessment of authorisation  

5.6.5.12.3.1 Norms and criteria  

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for textile-attacking insects in the following way: 

Products intended for use as repellent against textile-attacking insects 

Non-insecticidal  efficacy  has  to  be  proven  in  a  simulated-use  test  if  it  cannot  be  waived  (for 
details see Introduction, section 5.6.5.1.3.8). 

Laboratory tests (not required for product authorisation): 

•  ≥  80%  repellence  of  adults  and  larvae  within  the  test  period  according  to  the  claim 
(sex, developmental stage, type of tissue, etc.) and until the end of the residual period; 

•  ≤ 20% offspring compared to 100% of control group; 

 
 
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The required results for simulated-use tests are:  

Claim “protect textiles” or “prevents larvae from feeding” 

•  100% textile protection documented by photography (i.e. no damage on textiles in the 
test)  within  the  test  period  according  to  the  claim  and  until  the  end  of  the  residual 
period; 

•  ≥  80%  repellence  of  adults  and  larvae  within  the  test  period  according  to  the  claim 
(sex, developmental stage, type of tissue, etc.) and until the end of the residual period; 

• 

in the treated closets ≤ 20% offspring compared to 100% of control group. 

Claim “effective against textile-attacking insects” 

•  ≥  80%  repellence  of  adults  and  larvae  within  the  test  period  according  to  the  claim 
(sex, developmental stage, type of tissue, etc.) and until the end of the residual period; 

• 

in the treated closets ≤ 20% offspring compared to 100% of control group. 

Attractants without PT18 active substances 

The required results for simulated-use tests are:  

•  ≥ 80% attractiveness compared to the negative control within the test period according 
to  the  claim  (sex,  developmental  stage,  type  of  tissue,  etc.),  from  the  beginning  and 
until the end of the claimed efficacy period; 

• 

for sex specific attractants: ≥ 80% attractiveness for the receiving sex; 

•  at least a ratio of 4:1 of test individuals trapped in the trap with attractant compared to 
the control trap within the test period (or according to the claim), from the beginning 
and until the end of the claimed efficacy period. 

5.6.5.13 Ticks 

5.6.5.13.1 Introduction  

Ticks  (order:  Ixodida),  along  with  mites  belong  to  the  subclass  Acarina  (also  known  as  Acari) 
within  the  class  Arachnida.  All  ticks  are  obligatory  blood-sucking  parasites,  and  certain  tick 
species  can  carry  and  transmit  a  variety  of  different  pathogenic  microorganisms  including 
bacteria,  viruses,  protozoa  and  fungi.  The  most  important  tick  vector  species  in  Europe  is 
Ixodes  ricinus.  Diseases  vectored  by  this  tick  include  Lyme  disease  (also  known  as  Lyme 
borreliosis),  tick-borne  encephalitis  (TBE),  rickettsiosis,  babesiosis,  and  anaplasmosis,  which 
can  affect  both  humans  and  animals.  The  tick  Hyalomma  marginatum  is  a  vector  of  the 
Crimean-Congo  haemorrhagic  fever  virus  (CCHF),  in  Europe  mainly  restricted  to  the  eastern 
Mediterranean  area  (Turkey  and  the  Balkan  region),  Spain  and  the  southern  parts  of  France 
and  Italy.  The  Mediterranean  spotted  fever,  a  rickettsial  disease,  is  transmitted  by  the  brown 
dog  tick,  Rhipicephalus  sanguineus.  This  tick  occurs  mainly  in  buildings  where  dogs  live  and 
can  transmit  a  number  of  pathogens.  Further  abundant  species  are  Dermacentor  marginatus 
that  occurs  predominantly  in  the  Mediterranean  climate  zone,  and  Dermacentor  reticulatus, 
occurring in slightly cooler areas. The latter is a known vector of Babesia canis, causing canine 
babesiosis, and both can transmit rickettsia spp. 

PT19  biocides  against  ticks  can  only  claim  to  repel  or  attract  (in  order  to  trap)  the  target 
organisms, not to prevent the diseases. 

5.6.5.13.1.1 Biology  

Ticks  are  characterized  by  two  main  body  parts  and  eight  legs  as  nymphs  and  adults,  as 
opposed  to  insects,  which  have  three  main  body  parts  and  six  legs.  Ticks  go  through  four 
stages  to  complete  their  lifecycle:  egg,  six-legged  larva,  eight-legged  nymph,  and  adult. 
Feeding will occur in both the immature and adult stages. 

Except one African species (Nuttalliellia namaqua), all ticks can be classified into two families: 
hard  ticks  (Ixodidae)  and  soft  ticks  (Argasidae).  Key  differences  are  that  soft  ticks  lack  the 
sclerotized scutum (dorsal shield) apparent in hard ticks and that the mouthparts of soft ticks 
are  located  ventrally  and  cannot  be  seen  from  above,  in  contrast  to  hard  ticks  having  their 
mouthparts located distally, thus being easily visible from above. 

 
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There  are  also  differences  in  the  life  cycle:  most  soft  ticks  develop  through  several  nymphal 
instars  (usually  2-4),  and  the  adult  female  tick  can  repeatedly  feed  blood  (usually  2-5  times) 
and  typically  lay  between  100  and  300  eggs  after  each  blood  meal,  which  most  often  lasts 
between  20  and  50  minutes.  In  hard  ticks,  there  is  only  one  nymphal  instar  and  the  adult 
female  tick  only  feeds  once,  followed  by  oviposition  of  hundreds  or  up  to  thousands  of  eggs. 
The  blood  meal  of  hard  ticks  lasts  between  2  and  8  days,  depending  on  species  and  stage. 
During that time, the tick is fixed to the host`s body.  

There are two basic host-seeking strategies in ticks: the hunting and the ambushing strategy. 
Hunter ticks typically stay in their shelter until they start host-seeking. Then they actively crawl 
to and onto their hosts. Most soft ticks and also a number of nidicolous hard ticks belong to this 
group,  but  also  the  hard  ticks  R.  sanguineus  and  many  Hyalomma  species  including  H. 
marginatum.  Ambushing  ticks,  in  contrast,  seek  an  exposed  position  like  grass  or  shrubs  and 
wait  there  for  prolonged  periods  in  order  to  cling  to  a  passing  host.  Once  on  the  host’s  skin, 
ticks begin to crawl in search of a place to feed. 

Commonly, ticks attach to human skin along pant or sock lines or other tight locations that are 
warm and humid. 

Ticks can be differentiated according to their host preferences: 

• 

• 

• 

“One  host  ticks”:  developing  stages  and  adults  feed  on  the  same  host  individual,  e.g. 
Rhipicephalus microplus.  

“Two  hosts  ticks”:  larvae  and  nymphs  feed  on  the  same  host,  adults  feed  on  another 
host, e.g. H. marginatum.  

“Three hosts ticks”: larvae, nymphs and adults feed on three different host individuals, 
e.g. I. ricinus, D. reticulatus. 

5.6.5.13.2 Dossier requirements 

Dossier requirements are stated in the Introduction (see section 5.6.5.1.3). 

In order to eliminate the risk of disease transmission to human volunteers in field settings, field 
trials  with  repellents  against  ticks  are  not  required  for  authorisation  of  products  applied  on 
humans or clothing (for details see Introduction, section 5.6.5.1.3.4.3). In the case of products 
applied  to  domestic  animals,  field  trials  can  be  less  stressful  for  the  animals  and  should 
therefore be accepted instead of simulated-use trials. 

For authorisation of products applied on human or animal skin efficacy must be proven with the 
recommended  dose  and  according  to  the  claimed  mode  of  application  given  on  the  label  and 
SPC.  The  dose  used  in  the  efficacy  studies  should  be  covered  by  human  health  risk 
assessment. 

5.6.5.13.2.1 Test species 

Products  with  the  claim  “against  ticks”  applied  for  the  use  on  human  skin  or  for  the  use  as 
surface or spatial treatment has to be tested either on I. ricinus or on I. scapularis. Note that 
for some active substances,  efficacy may be different and then the use of the chosen species 
should  be  justified,  see  Appendix  18:  Büchel  K.,  et  al..  For  a  species-specific  claim,  testing 
against the claimed species is required. The efficacy of products applied on humans should be 
proven  against  adults  and  nymphs.  Depending  on  which  developmental  stage  parasitises  the 
host, the biological  relevant developmental stage must be tested (e.g. nymphs and adults  for 
Ixodes  ricinus  and  I.  scapularis;  adults  for  Dermacentor  reticulatus,  D.  marginatus  and 
Hyalomma  marginatum).  Therefore,  the  applicant  should  provide  information  and  justification 
on  the  developmental  stage  of  the  ticks  used  in  the  efficacy  test.  For  a  general  claim  against 
ticks, both Ixodes adults and nymphs have to  be tested,  otherwise, the tested developmental 
stage needs to be stated in the SPC and on the label claim. If CPTs with nymphs and adults are 
different, the shortest CPT should be stated on the SPC and label. 

For  a  general  claim  for  the  repellence  of  ticks  on  dogs  or  products  intended  for  surface 
treatment when dogs are present, at least two different tick species parasitising dogs should be 
tested  (R.  sanguineus  and  either  I.  ricinus  or  a  European  Dermacentor  species).  For  species 
specific  claims,  just  the  target  species  has  to  be  tested.  The  repellent  effect  for  products 
applied  on  domestic  animals  should  be  demonstrated  for  each  tick  species  and  each  target 
animal  species,  i.e.  dog,  horse,  etc.  claimed.  Dogs  should  be  used  as  a  substitute  for  cats, 

 
 
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however, as testing with cats can imply certain animal welfare issues. The efficacy of products 
used on domestic animals should be proven against adult ticks because testing with nymphs is 
not easily feasible even on short animal fur. 

There are further Rhipicephalus species difficult to differentiate from R. sanguineus. Therefore, 
the  species  of  the  test  organism  should  be  well-defined,  and  the  origin  of  the  organisms 
specified. 

When  use  in  tropical  areas  is  claimed,  Hyalomma  marginatum  or  Amblyomma  variegatum 
should also be tested. H. marginatum behaves differently than I. ricinus and I. scapularis, since 
it actively seeks the host to feed on and moves quickly on the ground.  

Due to the  specificity of certain active substances,  for products based on an active substance 
with a species-specific  mode  of action, only  effects against tick species that have been tested 
under simulated-use and/or field conditions, depending on the type of claim, should be claimed 
on the product label and in the SPC. 

Table 30: Overview of the required test species depending on the claim 

Type of product 

Claim 

Test species 

General claim 
against ticks 
Species-specific or 
developmental-
specific claim 

Ixodes ricinus or, 
Ixodes scapularis 

Claimed species  

Topical repellents 
on human skin, 
Surface repellents, 
Spatial repellents 

Use in tropical areas 

Topical repellents 
on domestic 
animals, 
Surface repellents 
in the area of 
domestic animals 

General claim 
against ticks 

Species-specific 
claims 

Ixodes ricinus, or 
Ixodes scapularis  
and 
either Hyalomma 
marginatum, or 
Amblyomma variegatum 
Ixodes ricinus and either 
Rhipicephalus 
sanguineus, or European 
Dermacentor species or 
Hyalomma species 

Developmental 
stage 

Nymphs and 
adults 
Claimed 
developmental 
stage 

Ixodes species: 
Nymphs and 
adults  

Others: Adults 

Adults 

Claimed species 

Adults 

Attractants 

Only species-specific 
claims possible 

Claimed species  

Claimed 
developmental 
stage 

5.6.5.13.2.2 Requirements per type of claim and test methods 

A laboratory test is not required for product authorisation but can be used to determine one or 
several  effective  concentrations  under  laboratory  conditions  and  to  see  whether  the  product 
sufficiently  repels/attracts  under  laboratory  conditions.  For  active  substance  approval  only,  a 
laboratory  test  is  sufficient.  However,  the  laboratory  test  can  be  waived  for  active  substance 
approval when a suitable simulated-use test is provided. 

5.6.5.13.2.2.1 Products intended for use as topical repellents for human skin 

The  efficacy  of  repellent  products  targeting  the  use  on  human  skin  should  be  proven  in  a 
simulated-use test according to the instructions for use (description see 5.6.5.13.2.2.1.2).  

In order to eliminate the risk of disease transmission to human volunteers in field settings, field 
trials  with  repellents  against  ticks  are  not  required  for  authorisation  of  products  applied  on 
human  skin  (for  details  see  Introduction,  chapter  5.6.5.1.3.3.3).  Pre-existing  studies  may  be 
submitted  as  additional  information.  If  field  trials  are  conducted,  they  must  take  place  in  an 
area  with  an  appropriate  tick  density  and  at  a  time  when  the  relevant  tick  species  are 
abundant.  These  tests  should  preferably  take  place  in  Europe  or  other  relevant  regions 
according  to  the  claims  (e.g.  tropical  regions).  Where  testing  in  Europe  is  not  possible,  the 
conditions  and  tick  species  must  be  confirmed,  and  their  relevance  justified.  Time  of  day  at 

 
 
 
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which  volunteers  are  treated  and  at  which  exposure  started  and  ended  should  be  reported 
along  with  the  weather  conditions  (temperature,  humidity,  precipitation,  wind  speed,  light 
intensity,  cloudiness).  At  least  10  volunteers  (preferably  an  equal  number  of  males  and 
females;  age:  18–65  years)  should  be  included  in  the  field  trial.  To  avoid  tick  bites  on 
volunteers  negative  controls  are  not  necessary,  because  field  trials  can  only  be  used  as 
supportive data. If field trials are conducted, care must be taken to avoid tick bites. 

The repellent products used for the test should be identical to the product to be marketed. 

CPT  values  must  be  determined  in  a  simulated-use  test.  As  field  studies  are  not  assessed  as 
key studies, data from the field studies cannot be used to determine CPT values.  

5.6.5.13.2.2.1.1 Laboratory test 

Different set-ups are recommended for testing repellent products targeting the use on human 
skin, see Appendix 18. Only one appropriate set-up is required per use. 

5.6.5.13.2.2.1.2 Simulated-use test 

Volunteers:  Efficacy  testing  should  involve  at  least  10  different  volunteers  (preferably 
volunteers with different hairiness of the arms, different genders; age: 18-65 years). For CPT 
calculation  valid  data  of  at  least  10  different  volunteers  must  be  used.  Volunteers  need  to  be 
fully  informed  about  the  aim,  the  procedure  and  the  expected  duration  of  a  study.  The  study 
should  be  carried  out  in  compliance  with  the  national  ethics  regulation.  They  also  need  to  be 
informed  about  potential  side  effects  such  as  allergic  skin  reactions  caused  by  the  product. 
Every  side  effect  observed  during  the  test  should  be  mentioned  in  the  test  report.  Their 
participation  is  voluntary  and  can  be  recalled  at  any  time  before  and  during  the  study,  see 
Appendix  18:  OPPTS  810.3700.  12  hours  before  and  during  testing,  volunteers  should  avoid 
nicotine, alcohol, fragrances (perfumes, body lotions,  soap,  etc.) and  repellent products. Prior 
to the application of the repellent, the skin is washed with fragrance-free soap and rinsed with 
water. 

Test  design:  The  repellent  product  is  applied  to  human  forearms  from  approximately  5  cm 
above the wrist to the elbow. As a negative control, an untreated arm of the same test person 
will be tested. This also serves to pre-screen ticks for sufficient crawling activity. The criteria of 
test  time  and  distance  in  the  control  should  correspond  to  the  test  run  with  the  repellent 
product. On the control arm, a line is drawn approximately 5 cm above the wrist to mark the 
beginning  of  the  crossing  zone.  To  mark  the  release  point  of  the  ticks  a  line  is  drawn  at  a 
distance  of  3  cm  below  the  crossing  zone  (see  Figure  15).  To  mark  the  end  of  the  crossing 
zone, a third line is drawn 3 cm above the line that marks the beginning of the crossing zone. 
In order to be considered as sufficiently locomotive, a tick needs to cross the crossing zone. On 
the treated arm, the same lines as above are drawn. Ticks showing sufficient crawling activity 
are used for the test on the treated arm immediately after the control test. 

Test animals must be active for host searching behaviour, which is after a starvation period of 
a minimum of 6 months (for Ixodes, for other species a shorter starvation period of 3 months 
is  possible)  after  the  last  blood  meal.  Ticks  must  be  pathogen-free.  The  arms  are  inverted  to 
promote upward movement since most ticks are negatively geotropic. Ticks are placed on the 
untreated release point either on the bottom or the upper side of the arm 3 cm below the test 
area with forceps or a brush. The side used must be documented in the study report. The same 
side of the arm must be used during the entire study to get comparable data. While  I. ricinus 
moves  upwards  by  themselves,  I.  scapularis  needs  to  be  gently  ‘guided’  upwards  using  a 
paintbrush  (see  Appendix  18:  Carroll  S.P.).  Each  tick  that  has  crossed  into  the  marked  3  cm 
zone  and  crossed  the  3  cm  zone  (tick  species  like  I.  ricinus)  or  stayed  there  for  1  min  (tick 
species  like  I.  scapularis,  which  is  a  species  with  low  locomotive  activity)  is  considered  as 
sufficiently locomotive and can be directly used as a test tick on the treated arm.  

Exposure  periods  should  take  place  every  30  or  60  minutes  with  the  first  being  30  or  60 
minutes  after  the  product  application.  Each  tick  should  be  observed  for  a  maximum  of  3 
minutes. 5 or 10 ticks should be tested within each 30 or 60 minutes intervals, respectively, by 
each volunteer, giving  a total of 10 ticks per  hour and volunteer. If two species are tested in 
one trial the same criteria are applicable, i.e. 10 ticks per hour and volunteers must be tested 
per species. For testing both nymphs and adults for a general claim against ticks 5 nymphs + 5 
adults (= 10 ticks) per hour and volunteers must be tested. For testing only one developmental 
stage 10 ticks (either 10 nymphs or 10 adults) per hour and volunteers must be tested. 

 
 
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Figure  15:  Upper  side  of  a  human  test  arm  in  a  simulated-use  test  to  show  repellency  against  ticks.  Dotted  lines 
(release point, start and end of the 3 cm crossing zone) mark the lines that are drawn on both the control and the test 
arm before the test. 

The following criteria for repellence should be used: 

A tick is not repelled when it, within the 3 minutes observation time, either  

•  Crawls onto the treated area and remains there for 1 min, or  

•  Enters  the  treated  area  and  further  crawls  across  the  3  cm  crossing  zone  (see  Figure 

15) within less than 1 min after entering the treated area. 

A tick is repelled when it, within the 3 min observation time, either 

•  does not crawl into the treated area, i.e. stays on the untreated area or drops off from 
the untreated area of the treated arm after contacting the start line of the crossing zone 
(repellent border), or 

• 

• 

crosses  the  start  line  of  the  crossing  zone  (repellent  border),  but  turns  back  into  the 
untreated area within less than 1 min after entering the treated area, or 

crosses the start line of the crossing zone but drops off from the treated area within less 
than 1 min after entering the treated area. 

A tick that does not move at all on the test arm, even after moving on the control arm, has to 
be excluded from the test, and another tick should be used instead. Every tick should only be 
used once for a test to avoid habituation effects. When a tick starts to bite, this tick should be 
removed directly from the skin. When a tick starts to bite hereby on the treated area this tick is 
considered as non-repelled. Biting can be prevented by constant observation. 

Efficacy assessment:  While testing for repellence, an endpoint  for the failure  of  repellence for 
subjects  treated  with  the  recommended  dose  should  be  selected.  Efficacy  failure  in  a  test  to 
determine  CPT  is  the  time  from  the  application  of  a  repellent  until  efficacy  failure  by  a 
confirmed event (definition see Introduction, section 5.6.5.1.5). Repellent effectiveness should 
be based on the median or mean CPT (CPT calculation see Introduction, section 5.6.5.1.5). 

The claimed CPT for a general claim should correspond to the shortest CPT (mean or median) 
among  all  tested  species  and  should  be  stated  on  the  label  and  SPC.  For  specific  claims,  the 
mean or median for each species respectively may be stated separately on the label and SPC. 

 
 
 
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If different developmental stages have been tested, the shortest CPT (mean or median) should 
be indicated on the label and SPC. 

Proof  of  non-insecticidal  efficacy,  if  it  cannot  be  waived  (for  details  see  Introduction,  section 
5.6.5.1.3.8):  The  same  test  setup  as  described  above  can  be  used  with  a  defined  number  of 
ticks (at least 50 individuals, with 5 individuals per volunteer). Ticks are placed in the treated 
area  on  the  line  that  marks  the  end  of  the  crossing  zone.  The  arms  are  inverted  to  promote 
upward  movement  since  most  ticks  are  negatively  geotropic.  To  simulate  a  natural  situation, 
ticks should be removed after one minute if they do not leave the treated area by themselves. 
Afterwards  the  ticks  should  be  kept  separately  in  the  laboratory  and  mortality  should  be 
recorded 24 hours after exposure. This test should be conducted within the 30 minutes directly 
after the product application and before the first exposure of ticks for repellency testing.  

5.6.5.13.2.2.2 Repellents applied on clothing both for humans or animals 

For  product  authorisation  purposes  the  efficacy  of  repellent  products  applied  on  clothing  for 
humans  should  be  proven  in  a  simulated-use  test  according  to  the  instructions  for  use 
(description see 5.6.5.13.2.2.2.1). 

CPT  values  must  be  determined  in  a  simulated-use  test.  As  field  studies  are  not  assessed  as 
key studies, data from the field studies cannot be used to determine CPT values. 

In order to eliminate the risk of disease transmission to human volunteers in field settings, field 
trials  with  repellents  against  ticks  are  not  required  for  authorisation  of  products  used  on 
humans  (for  details  see  Introduction,  section  5.6.5.1.3.4.3).  Pre-existing  studies  may  be 
submitted  as  additional  information.  If  field  trials  are  conducted,  they  must  take  place  in  an 
area  with  an  appropriate  tick  density  and  at  a  time  when  the  relevant  tick  species  are 
abundant.  These  tests  should  preferably  take  place  in  Europe  or  other  relevant  regions 
according  to  the  claims  (e.g.  tropical  regions).  Where  testing  in  Europe  is  not  possible  the 
conditions  and  tick  species  must  be  confirmed,  and  their  relevance  justified.  Time  of  day  at 
which  volunteers  are  treated  and  at  which  exposure  started  and  ended  should  be  reported 
along  with  the  weather  conditions  (temperature,  humidity,  precipitation,  wind  speed,  light 
intensity,  cloudiness).  At  least  10  volunteers  (preferably  volunteers  with  different  hairiness  of 
the  arms,  different  genders;  age:  18–65  years)  should  be  included  in  the  field  trial.  To  avoid 
tick  bites  on  human  volunteers  negative  controls  are  not  necessary,  because  field  trials  can 
only be used as supportive data. If field trials are conducted, care must be taken to avoid tick 
bites. 

For  product  authorisation  purposes  the  efficacy  of  repellent  products  applied  on  clothing  for 
animals should be proven in: 

•  a simulated-use test according to the instructions for use, or 

•  a field test according to the instructions for use.  

The repellents used for the test should be identical to the product to be marketed. 

Proposed claims regarding the protection of a specific type of fabric must be simulated, i.e. the 
same type of fabric has to be used in the simulated-use test. 

Proposed  claims  regarding  the  performance  of  the  product  need  to  be  simulated  in  the  study 
(for details see Introduction, section 5.6.5.1.3.5).  

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  substrates/surfaces  or  repellent  product  (temperature,  humidity,  photoperiod, 
ventilation). 

If  outdoor  use  is  claimed,  the  treated  surface/substrate  or  repellent  product  and  control 
surfaces/substrates  have  to  be  kept  outside  to  ensure  adequate  weathering  and  the  outdoor 
conditions must be specified (for details see Introduction, section 5.6.5.1.4.1.3). 

The  efficacy data should  be  relevant to prove  the submitted  claims. Therefore, the  efficacy of 
biocidal products that are limited to a body area, e.g. collars must be proven for the whole test 
individual  that  should  be  protected.  If  only  specific  body  areas  intend  to  be  protected,  e.g. 
ears, udders, etc. then the efficacy for the claimed area must be proven. 

For specific claims (prevention of bites through/prevention of bites next to the treated clothes), 
relevant tests have to be submitted. 

 
 
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5.6.5.13.2.2.2.1 Simulated-use test 

The efficacy of products intended for use on clothes or fabrics for humans should be proven in 
a similar test as described in  section 5.6.5.13.2.2.1.2 by covering the test arm with a treated 
cloth and the control arm with an untreated cloth. The fabric used has to be representative to 
the  fabrics  commercially  used,  i.e.  thick  uniform  cotton  if  the  field  of  use  is  military  clothing, 
etc. The ticks should be released in an untreated area, e.g. fabric tape on the textile, allowing 
the tick a barrier-free transition to clothing. 

For  efficacy  assessment  and  determination  of  non-insecticidal  efficacy  please  see 
5.6.5.13.2.2.1.2. 

5.6.5.13.2.2.3 Repellent products intended for use as spatial treatment 

For product authorisation purposes the efficacy of repellent products intended for use as spatial 
treatment should be proven in: 

•  a simulated-use test according to the instructions for use, or  

•  a field test according to the instructions for use. 

These  kinds  of  products  are  not  sought  for  the  application  to  humans  or  animals.  Therefore, 
field  studies  can  be  conducted  without  human  or  animal  probands  and  the  risk  of  vector 
transmitted diseases can be neglected.  

The repellents used for the test should be identical to the product to be marketed. 

Replicates:  A  minimum  of  5  independent  replicates  (each  treatment  and  negative  control) 
should be performed. The pre-treatment counts of ticks or control plots can serve as negative 
controls. 

For a general claim “spatial repellents” the repellent effect must be proven. For a specific claim 
“dispelling” the dispelling effect must be proven. 

For residual efficacy, ticks are exposed to the product at several time intervals after application 
(including the end of the claimed period). 

The  room  size  stated  in  the  SPC  and  on  the  label  must  be  used  in  the  efficacy  trial.  For  the 
extrapolation to larger room sizes than proven in the efficacy test a justification is necessary. 

If  outdoor  use  is  claimed,  the  test  should  be  performed  outdoor  and  the  treated 
surface/substrate or repellent product and control surfaces/substrates have to be kept outside 
to  ensure  adequate  weathering  and  the  outdoor  conditions  must  be  specified  (for  detailed 
information regarding the requirements see Introduction, section 5.6.5.1.4.1.3). 

Proof  of  non-insecticidal  efficacy,  if  it  cannot  be  waived  (for  details  see  Introduction,  section 
5.6.5.1.3.8) have to be examined at the end of the trial. 

5.6.5.13.2.2.4 Repellent products intended for use as surface treatment 

For product authorisation purposes the efficacy of repellent products should be proven in: 

•  a simulated-use test according to the instructions for use, or 

•  a field trial according to the instructions for use and additionally a laboratory trial testing 

the required different surfaces. 

These  kinds  of  products  are  not  sought  for  the  application  to  humans  or  animals.  Therefore, 
field  studies  can  be  conducted  without  human  or  animal  probands  and  the  risk  of  vector 
transmitted diseases can be neglected.  

Products applied onto surfaces may act either by evaporation or on the surface itself.  

For products applied on surfaces, two porous and one non-porous surface should be used, e.g. 
ceramic  tile,  plywood,  painted  plywood,  stainless  steel,  concrete,  for  a  general  claim  as 
“surface treatment”. The efficacy of each surface should be proven in a separate test, i.e. three 
tests for three different surfaces. For authorisation of a product to be used on a specific type of 
surface the efficacy for only this specific surface should be assessed. 

Replicates: A minimum of 5 independent  replicates should be performed (each treatment and 
negative control). 

 
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For residual efficacy, ticks are exposed to the product at several time intervals after application 
(including the end of the claimed period). 

If  outdoor  use  is  claimed,  the  treated  surface/substrate  or  repellent  product  and  control 
surfaces/substrates  have  to  be  kept  outside  to  ensure  adequate  weathering  and  the  outdoor 
conditions  must  be  specified  (for  detailed  information  regarding  the  requirements  see 
Introduction, section 5.6.5.1.4.1.3). 

Proposed  claims  regarding  the  performance  of  the  product  should  be  simulated  in  the  study. 
For  example,  for  the  claim  “unaffected  by  cleaning/vacuuming”,  the  surface  should  be 
repeatedly cleaned during the trial (for details see Introduction, section 5.6.5.1.3).  

Proof  of non-insecticidal  efficacy, if it  cannot be waived, have to be examined in a simulated-
use test (for details see Introduction, section 5.6.5.1.3.8). 

5.6.5.13.2.2.5 Repellents applied on animals 

For product authorisation purposes the efficacy of repellent products applied on animals should 
be proven in: 

•  a  simulated-use  test  according  to  the  instructions  for  use  (test  design  example  see 

5.6.5.13.2.2.5.1), or 

•  a field test according to the instructions for use.  

Due  to  practical  reasons,  tests  on  animals  should  be  performed  with  adult  ticks,  as  nymphs 
might  be  more  difficult  to  handle  on  animal  fur  and  as  the  adult  stage  is  the  most  common 
stage found on companion animals like cats or dogs.  

Topical  repellents  for  animals  should  be  tested  against  the  targeted  tick  species  and  on  the 
animal,  e.g.  dog,  horse,  etc.  that  shall  be  protected.  Dogs  should  be  used  as  a  substitute  for 
cats, as testing with cats can imply certain animal welfare issues.  

The repellents used for the test should be identical to the product to be marketed. 

Test animals: It is recommended to include at least 10 animals per treatment/control group of 
different  breed  and  sex,  i.e.  for  testing  on  entire  animals  at  least  10  treated  and  10  control 
individuals  are  necessary  since  repellence/attractiveness  to  ticks  varies  considerably  between 
animal individuals.  

Depending  on  the  animal  species,  several  factors,  e.g.  hair  length,  the  thickness  of  the  coat, 
self-grooming, etc. might impact the efficacy. These factors should be taken into account in the 
demonstration  of  the  efficacy.  The  two  groups  should  be  kept  separately,  to  avoid  contact  of 
the control animals with the treated fur. 

The  product  should  be  applied  according  to  the  claim.  Claimed  application  rates  should  take 
into account the type and weight of the animals. The dose used in the efficacy studies should 
be covered by risk assessment. 

It  is  necessary  that  animals  come  from  suitable,  e.g.  non-smoking  households.  Every  side 
effect  observed  during  the  test  should  be  mentioned  in  the  test  report.  24  hours  before  and 
during  testing,  animals  should  not  be  treated  with  fragrance  (perfumes,  soap,  etc.).  The  test 
animal  should  not  be  protected  by  any  previous  repellent  and/or  insecticidal  residuals  caused 
by  previous  treatment.  Concerning  endo-antiparasitic  agents  this  is  acceptable,  but  details 
(product,  dose,  application  rate,  application  method,  date  of  the  last  treatment,  and  residual 
efficacy)  must  be  given  in  the  report  and  the  controls  and  the  treated  groups  must  have  the 
same  status.  A  safety  margin  for  externally  applied  anthelmintics  is  4  weeks  after  the  end  of 
the  last  application.  The  history  of  the  treated  and  the  control  groups  should  be  comparable. 
The status of the animals should be specified and only animals for which an insecticidal effect 
can  be  excluded  should  be  included  in  the  study.  Due  to  feasibility  and  cost-effectiveness,  it 
would  be  possible  to  use  10  animals  one  week  (5  each  in  the  treatment  and  control  groups) 
and  then  use  the  same  individuals  again  in  the  second  week,  but  swap  groups.  Each  animal 
would  then  act  as  its  own  control.  However,  any  residual  activity  of  the  product  has  to  be 
excluded. 

The investigator is reminded that the validity of the results is directly related to the degree of 
variability in the test. Increasing the number of test animals could increase the reliability of the 
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Proposed  claims  regarding  the  performance  of  the  product  should  be  simulated  in  the  study. 
For  the  claim  “unaffected  by  washing”,  the  label  and  the  SPC  must  indicate  how  often  the 
animal can be washed without reducing the efficacy of the biocidal product. In the efficacy test, 
the  test  individuals  should  be  repeatedly  washed  with  a  non-insecticidal,  fragrance-free 
shampoo during the trial according to the number of wash cycles  indicated. If a product claim 
is to protect the entire animal, this should be demonstrated (notably for collars). 

5.6.5.13.2.2.5.1 Simulated-use test 

The candidate repellent is applied according to the instructions for use. If the product is acting 
at very close range it may be possible to make the evaluation by treating parts of the host, like 
one treated side of the neck compared with the equivalent area on the other, untreated, side of 
the neck. The advantage  of  such a setup is the neutralisation  of individual host differences in 
tick-attraction. The control body part should resemble the treated body part (e.g. front-half vs 
back-half or left side vs right side of the animal). In case of a restricted application, e.g. collar, 
a spot-on product with a claim of entire body protection or for products acting in a wider range 
over the entire body the test should permit to validate the efficacy of this kind of application by 
testing  the  entire  animal.  In  this  case  untreated  animals  must  be  used  as  a  control.  The 
negative controls serve to pre-screen ticks for sufficient locomotive activity.  

The  attractiveness  of  the  animal  should  be  validated  before  the  trial.  Animal’s  attractiveness 
can be measured by exposing the untreated skin to ticks.  

The detailed measurements will depend on the size of the animal, and the following dimensions 
adapted  from  the  simulated-use  test  on  humans  should  be  used  only  as  guidance.  Other  test 
designs than the following examples can be accepted if the protocol is scientifically valid. 

Test  design:  On  the  control  leg  as  well  as  on  the  treated  leg,  a  3  cm  width  crossing  zone  is 
marked  (e.g.  by  drawing  or  shaving  a  line)  approximately  5  cm  above  the  carpal  joint.  The 
release point of the ticks is marked by a line at a distance of 3 cm below the crossing zone. In 
order to be considered as sufficiently locomotive, a tick needs to cross the crossing zone.  

Alternatively,  products  can  be  applied  according  to  the  label  claim  and  SPC  on  one  side  (= 
treated side) of the test animal, e.g. the lateral area of the thorax. The other side of the test 
animal should be used as negative control to evaluate sufficiently locomotive ticks. During the 
observation the animals are allowed to  stand, sit or lay down. The ticks can be placed on the 
fur of the treated side on an untreated sleeve. The ticks must be released on an untreated area 
of at least 1 cm width. In order to be considered as sufficiently locomotive, a tick needs to walk 
on the untreated side into the fur to the skin within 3 minutes observation time. Biting can be 
prevented by constant observation. 

Ticks  must  be  active  for  host  searching  behaviour,  which  is  after  a  starvation  period  of  a 
minimum of 6 month (for Ixodes, for other species a shorter starvation period of 3 months is 
possible)  after  the  last  blood  meal  and  must  be  pathogen-free.  Ticks  are  placed  on  the 
untreated release point 3 cm below the crossing zone with a forceps or brush. While  I. ricinus 
moves  upwards  by  themselves,  I.  scapularis  needs  to  be  gently  ‘guided’  upwards  using  a 
paintbrush (see Appendix 18: Carroll S.P.). Each tick that has crossed  into the marked 3 cm-
zone and crossed the 3 cm-zone (high locomotive tick species like I. ricinus) or stayed there for 
1 min  (low  locomotive  tick  species  like  I.  scapularis)  is  considered  as  sufficiently  locomotive 
and can be directly used as test tick on the treated leg. 

Alternatively, a tick that has walked on the control side into the fur to the skin within 3 minutes 
observation time is considered as sufficiently locomotive and can be directly used as a test tick 
on the treated other side of the thorax. 

Exposure  periods  should  take  place  every  30  or  60  minutes  with  the  first  being  30  or  60 
minutes  after  the  product  application.  Each  tick  should  be  observed  for  a  maximum  of  3 
minutes.  5  or  10  ticks  should  be  tested  within  each  30  or  60  min  intervals,  respectively,  by 
each test animal, giving a total of 10 ticks per hour and test animal. If two species are being 
tested in one trial the same criteria are applicable, i.e. 10 ticks per hour and test animal must 
be tested per species.  

The following criteria for repellence can be used:  

A tick is not repelled when it, within the 3 minutes observation time, either 

• 

crawls onto the treated area and remains there for 1 minute, or  

 
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•  enters  the  treated  area  and  further  crawls  across  the  3  cm  crossing  zone  within  less 

than 1 minute after entering the treated area, or 

• 

crawls onto the treated area from the fur to the skin. 

A tick is repelled when it, within the 3 minutes observation time, either 

•  does not crawl into the treated area, i.e. for the test design with marked areas the tick 
stays  on  the  untreated  area  or  drops  off  from  the  untreated  area  of  the  treated  area 
after contacting the repellent border (start line of the crossing zone), or 

• 

• 

crosses  the  start  line  of  the  crossing  zone  (repellent  border),  but  turns  back  into  the 
untreated area within less than 1 minute after entering the treated area, or 

crosses the start line of the crossing zone but drops off from the treated area within less 
than 1 minute after entering the treated area. 

A tick that does not move at all on the test area, even after moving on the untreated negative 
control area, has to be excluded from the test, and another tick should be used instead. Every 
tick should only be used once for a test to avoid habituation effects.  

For efficacy assessment and proof of non-insecticidal efficacy, if it cannot be waived, please see 
5.6.5.13.2.2.1.2. 

The investigator is reminded that the validity of the results is directly related to the degree of 
variability  in  the  test.  Increasing  number  of  test  animals  could  increase  the  reliability  of  the 
test results. 

Authorisation of products applied with a dose independent of animal size can be granted up to 
the bodyweight of the heaviest test animal. 

The claimed CPT for a general claim should correspond to the shortest CPT (median or mean) 
among  all  tested  species  and  should  be  stated  on  the  label  and  SPC.  For  specific  claims  the 
mean or median for each species respectively may be stated separately on the label and SPC. 

Environmental  conditions  must  be  specified  for  the  test  itself  (temperature,  humidity,  light 
intensity). 

5.6.5.13.2.2.6 Attractants without PT18 active substances  

For product authorisation purposes the efficacy of attractants in traps should be proven in: 

•  a simulated-use test according to the instructions for use, or  

•  a field test according to the instructions for use.  

Any attractant should be tested against a negative control. 

The  attractants,  e.g.  the  traps,  used  for  the  test  should  be  identical  to  the  product  to  be 
marketed (for details see Introduction, section 5.6.5.1.3.7). Any bycatch of non-target insects 
has to be recorded and identified at least to order, preferably to the family level. 

Environmental  conditions  must  be  specified  for  the  test  itself  and  during  the  storage  of  the 
treated  substrates/surfaces  or  attractant  product  (temperature,  humidity,  photoperiod, 
ventilation). 

If outdoor use is claimed, the attractant product and control have to be kept outside to ensure 
adequate  weathering  and  the  outdoor  conditions  must  be  specified  (for  detailed  information 
regarding the requirements see Introduction, section 5.6.5.1.4.1.3.). 

Replicates: A minimum of 5 independent  replicates should be performed (each treatment and 
negative control).  

In field trials the tick population can be determined by flagging of the vegetation.  

5.6.5.13.2.2.7 Attractants in PT18 bait products 

The  efficacy  evaluation  of  such  products  should  be  done  on  the  basis  of  the  requirements  for 
PT18  products  (see  PT18  Chapter:  Insecticides,  acaricides  and  products  to  control  other 
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5.6.5.13.3 Assessment of authorisation  

5.6.5.13.3.1 Norms and criteria  

According  to  the  BPR,  a  biocidal  product  may  only  be  authorised  if  it  “possesses  a  sufficient 
level of efficacy”. This is implemented for ticks in the following way: 

For  repellent  products  non-insecticidal  efficacy  has  to  be  proven  in  a  simulated-use  test  if  it 
cannot be waived (for details see Introduction, section 5.6.5.1.3.8). 

Products intended for use as repellent for human and animal skin, clothing 

The required results for simulated-use tests and field tests are:  

•  during the claimed protection period complete protection should be proven expressed as 

mean or median CPT (for more details see Introduction, section 5.6.5.1.5) 

•  CPT should correspond to the shortest CPT (mean or median) among the tested species 

and developmental stages 

Products intended for use as repellent as spatial or surface treatment 

The required results for laboratory tests, simulated-use tests and field tests are:  

•  during the claimed protection period ≥ 80% repellency; 

•  Protection period should correspond to the shortest period among the tested species. 

Products intended for use as attractants without PT18 active substance 

The required results for the different trials are: 

•  at least a ratio of 4:1 of test individuals trapped in the trap with attractant compared to 
the control trap within the test period (or according to the claim), from the beginning 
and until the end of the claimed efficacy period. 

Laboratory test or simulated-use test: 

•  ≥ 80% trapped test individuals compared to the negative control within the test period 
(or according to the claim), from the beginning and until the end of the claimed efficacy 
period. 

Field trial: 

•  ≥ 80% trapped test individuals compared to the negative control within the test period 
(or according to the claim), from the beginning and until the end of the claimed efficacy 
period. 

5.6.5.14 Wasps 

NOTE to the reader:  

This  section  of  the  guidance  contains  limited  information  concerning  repellents  against 
wasps included previously in chapter 5.6.4 and will be updated in the future in light of the 
experience  gained.  The  applicant  should  provide  a  testing  proposal  which  needs  to  be 
agreed upon by the respective CA in advance on a case-by-case basis. 

5.6.5.14.1 Test species 

A product for use against wasps should be tested on colonies and/or workers of Vespula spp. or 
Dolichovespula spp. 

5.6.5.14.2 Requirements per type of claim and test methods 

For  products  with  a  repellent  or  attracting  effect  against  wasps  no  agreed  protocols  are 
available. The tests should be designed to mimic the practical use situation.  The study results 
should provide a clear picture of the efficacy of the product. Methods should be described well. 
The submitted data from studies are checked for completeness, based on the applied dose per 
treated  area.  It  is  also  checked  whether  the  duration  of  exposure  is  sufficient.  If  the 
formulation  alone  i.e.  without  the  carrier,  e.g.  a  product  with  a  tissue  as  carrier,  has  been 
tested, data on release from the carrier are also required. 

 
 
 
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Products intended for repelling wasps 

• 

simulated-use or field trials. 

5.6.5.14.3 Assessment of authorisation 

5.6.5.14.3.1 Norms and criteria 

Products intended for repelling wasps 

• 

required results in a simulated-use or field test: 

-  a simulated-use test showing repellence; 

-  depending on the claim field test showing repellence. 

5.6.6 PT20 Other vertebrates 

Please refer to the General sections 1-3 of this guidance and the TNsG. 

5.7 Other biocidal products (Main group 4) 

5.7.1 PT21 Antifouling products 

5.7.1.1 General Introduction  

This  section  deals  with  the  methodology  for  the  evaluation  of  efficacy  tests  for  antifouling 
products  that  is  applicable  for  the  authorisation  of  products  under  the  EU  Biocidal  Products 
Regulation (BPR, Regulation (EU) 528/2012). 

5.7.1.1.1 Introduction 

This chapter describes the nature and extent of data which should be available to support the 
label claims for biocidal products within Product Type 21 - Antifouling Products. These are 
defined in the BPR as “Products used to control the growth and settlement of fouling organisms 
(microbes and higher forms of plant or animal species) on vessels, aquaculture equipment, or 
other structures used in water”. 

5.7.1.1.2 Types of Coating 

The antifouling products currently available can be categorised into the following broad coating 
types:  

•  Soluble matrix  

•  Insoluble matrix 

•  Self polishing  

The categorisation of coating types outlined above is general. It should be noted that some 
antifouling products do not necessarily rely on one single coating technology and combinations 
of different technologies have been developed by antifouling formulators to suit customer 
specifications and environmental requirements. A description of the main coating types can be 
found in Appendix 20. 

It should be noted that the protection periods described in the appendix for each coating type 
are typical life times that may be achieved by using products within these very broad groups. 
The efficacy of an antifouling coating will heavily depend upon use, for instance a vessel's 
operational pattern (such as dry-docking interval, sailing speed, and idle times as well as the 
temperature, fouling intensity, and other environmental characteristics where the vessel is 
trading). It also depends on the extent to which the antifouling paint specification has been 
tailored to meet these specific conditions. Surface preparation, primers, quality of work, dry 
film thickness, etc. may also affect the quality and/or duration of the protection. 

5.7.1.1.3 Mode of Action 

Antifouling products form paint films that act as release vehicles for the active substance(s) 
contained in the paints. The active substance(s) will be released over the specified lifetime of 
the products, creating a microlayer of biocide rich water at the paint surface. Here, in this 
water microlayer, the concentration should be sufficient to deter the settlement and/or growth 

 
 
 
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of fouling organisms. A more detailed description of the respective modes of action and 
physical characteristics of the various coating types are outlined in Appendix 20 of this 
document. 

5.7.1.1.4 Categorisation of antifouling products 

Antifouling paints are made available for different use types. Typically they are prescribed for 
yachts, commercial vessels (such as bulk carriers, tankers, container ships, car carriers, 
passenger ships, etc.), and aquaculture. 

The three broad categories of products (in Appendix 20) can be defined by the way in which 
the products control the release the active substance(s). Given the fact that a single active 
substance may not have a sufficiently broad spectrum activity to control the wide range of 
fouling organisms, antifouling products often contain more than one active substance. 

5.7.1.1.5 Spectrum of activity 

Target organisms belong to very different taxonomic groups. There are many organisms that 
can live within a fouling community, but only a few cause severe fouling problems. Which 
organisms will present a problem depends on the local conditions and the operation of the 
individual vessel. For example, typical target organisms in European waters may include, but 
are not limited to, various species of the following genus: Pseudomonas (light slime), Amphora 
(dense slime), Ulva (macro-algae), and Semibalanus (animals). 

Fouling organisms and growth rates differ between tropical and temperate regions. The fouling 
intensity and the species that dominate a fouling community may vary locally and seasonally. 
While it is not normally feasible to claim efficacy against specific target organisms, applicants 
may choose to supplement their label claim that the product is an ‘antifouling product’ with an 
indication as to whether the product will be effective against one or more of the following 
fouling groups: 

•  Slime 

•  Weed (macro-algae) 

•  Animals  

5.7.1.1.6 Dossier requirements 

The following aspects are required for the efficacy evaluation of antifouling products: 

1.  The label claims and instructions for use including the technical data sheet  

2.  Efficacy data on the product 

5.7.1.1.7 Label claims 

For each product a set of label claims should be provided as part of the dossier submitted. 
Claims for the activity of the product include those made on a technical data sheet or other 
associated documentation, as well as those on the label itself. To simplify the text, only the 
term 'label claim' will be used below. 

In general the claim for antifouling products can be rather unspecific, for instance 'antifouling 
product for professional application'. The label should also indicate to which fouling groups (see 
5.7.1.1.5) the product is effective and whether it can be used in marine or fresh water. 

The label claim for products used in areas other than on vessels, such as products used for 
aquaculture, in the inlet and outflow pipes of cooling systems, or for other “non-vessel” uses 
should be more precise, and clearly describe purposes for which the product can be used. 

According to Article 69(2)(f) of the BPR the label must clearly and indelibly show the uses for 
which a biocidal product is authorised. 

5.7.1.1.7.1 Areas Of Use 

The product label, technical data sheet or other associated documentation should contain 
information on the main use categories for the product, for example use on vessels and larger 
boats, yachts, stationary installations, or aquaculture equipment, etc. This will normally also 
include information on whether the product is intended (primarily or exclusively) for use in 
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As the fouling challenge is more severe under static conditions, installations and recreational 
boats (which are normally tied up in marinas) will foul more quickly than commercial vessels 
that spend most of their time in motion. Therefore, if a product is intended specifically for static 
or recreational use, this should be specified in the label claims. 

(For human risk assessment purposes, it is important that a label claim specifies if a product is 
intended for amateur use or if is for application by professionals only.) 

5.7.1.1.7.2 Application method/dose rate 

Antifouling coatings may be applied using methods such as airless and conventional spray, 
brush and roller, or dipping and immersion (aquaculture). The specified total dry film thickness 
will vary depending on the intended dry-docking interval, activity of the vessel (such as sailing 
speed and idle times), and on the temperature, fouling intensity, and other environmental 
characteristics where the vessel is operating. Furthermore, larger vessels will normally have 
different antifouling products and different paint film thicknesses specified for different parts of 
the underwater hull depending on, for instance, water flow and light conditions. Some areas, 
such as those with less frequent maintenance intervals than those for the rest of the 
underwater hull, and those with strong water throughput (e.g. inside thrusters) may require 
higher film thicknesses to minimize the risk of transmigration of non-indigenous species in 
these areas. 

It is important to note that the paint thickness does not affect the efficacy of a product, which 
will control fouling regardless of the thickness of the paint applied. Instead, the film thickness 
will define the in-service life of the product. 

For antifouling paints there is no direct relationship between the applied dose (paint film 
thickness applied) and the efficacy of the product (unlike agrochemicals, for example, where 
applying more pesticide increases the concentration of the pesticide and therefore the 
magnitude of the controlling effect on the pest). 

Recommended dry film thicknesses are given to ensure that enough paint is applied to the 
vessel to avoid the coating being ‘polished through‘ during service, exposing the underlying 
anticorrosive paint which will be susceptible to fouling. When paint is applied by spray, more 
than one coat of paint is normally applied to protect against possible application defects, such 
as ‘pin holing’, where small areas of the anticorrosive are left exposed. 

As the three major types of antifouling coatings (Appendix 20) vary in their ability to maintain 
a sufficient release of active(s), this is reflected in their different typical lifetimes. 

5.7.1.1.8 Efficacy tests 

5.7.1.1.8.1 Laboratory tests (including in-vitro screening tests) 

Laboratory tests are typically conducted on a single active substance and with a limited number 
of test organisms, and may provide information about the specific action of a substance against 
a known fouling species. It is acknowledged that model target organisms may be used in these 
tests as well as those that may successfully be cultivated in a laboratory (e.g. juvenile 
barnacles). Consideration should be given to the use of species known to be critical fouling 
species. 

Laboratory tests are routinely used to demonstrate efficacy of an individual active substance, 
often at a very early stage during research in order to screen new active substances. 

Laboratory testing of individual paints is not undertaken as it is not considered to be a realistic 
evaluation of the product. Field testing is routinely undertaken instead (described below). 

5.7.1.1.8.2 Simulated field tests (static raft testing) 

These may be studies that are conducted with the candidate product or with the active 
substance(s) incorporated into a model coating type. Such tests involve the immersion of 
panels treated with the test coating on static rafts for a period of months or years at an 
appropriate location. For aquaculture products this could be nets or (sections of) cages treated 
with the test product and immersed at an appropriate site. 

Efficacy data on antifouling coatings should normally be generated by testing over at least six 
months of peak fouling activity. As far as is practical the test location(s) should be 
representative of the intended uses of the product. When testing in locations with seasonal 

 
 
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variation in fouling challenge, the test period should cover the full fouling season. The length of 
a season will vary depending on the location of the test site. When choosing the test 
location(s), factors such as shelter (from strong waves and ship traffic) and access have to be 
balanced against water exchange conditions and other characteristics determining whether the 
water at a site is representative for the end use conditions. 

Since raft testing is carried out in natural environments, the same product may perform 
differently at the same site in different years. This variability in fouling intensity, and thus the 
test results, is due to weather conditions, availability of nutrients, and other uncontrollable 
factors that may affect the type and extent of fouling and its rate of settlement and growth. 
Therefore, a negative control (a surface which has no antifouling effect) should be included in 
all tests, which will indicate the degree of fouling that would be present under static conditions 
if the tested coatings were totally ineffective. A reference coating of proven or known efficacy 
(a positive control) may also be used. The absolute amount of fouling present on a test coating 
may not be reproducible at the same site from year to year. 

Efficacy studies include regular assessments of fouling throughout the period. These 
assessments usually describe the major types of fouling (e.g. slime, algae and other weeds, 
and barnacles or other fouling organisms), but describing these as to genus and species is 
unnecessary. As sharp edges on test panels may be difficult to protect, fouling that is not 
growing on the front of panels (i.e. attached along the edges) should be disregarded. 

The presentation of data should include the assessment method (the rating/scoring for the test 
panels and how these are interpreted), together with photographs and/or diagrams of the test 
panels. 

5.7.1.1.8.3 Field tests/In-service monitoring 

Since field tests involve long-term exposure to practical conditions, they can be regarded as in-
service tests. Field tests permit antifouling products to be tested under similar operating 
conditions and stresses as those encountered when the antifouling product are in service. 
Possible examples of these tests include: 

•  Panel tests where coated panels have been attached to a vessel during parts of or 

during a complete dry-docking interval 

•  Patch tests where vessels have been painted with the test coating as a strip or patch 

on the hull 

•  In-service monitoring of aquaculture nets, cages, etc. 

Any field data generated in support of an application should be conducted on the candidate 
product or representative products that closely resemble the fully formulated commercial 
product. A robust justification should be provided to support bridging of data from a similar 
(but not identical) product. 

It is recognised that it may not be possible to run concurrent untreated panels or patches 
during field trials. Therefore information on the performance of the main antifouling coating 
over the test period should be provided instead. Monitoring reports of the performance of an 
antifouling product on a fully treated vessel may also be submitted, where these are available. 
It is also recognised that data generation from field trials may require many years to carry out 
and are more likely to be available for well-known technologies than for products containing 
new active substances (or new combinations or concentrations of active substances) or for 
coating types based on new technologies. 

Where field data are not available, the applicant has the option to provide data on other 
existing formulation(s) where appropriate, and read across to the current application through 
scientific reasoned cases and arguments. Such arguments may include: 

•  The composition of the 'old' (and well documented) and the 'new' antifouling product  

•  Simulated field tests of the ‘old’ and the ‘new’ antifouling product 

•  Possible field data on the 'old' antifouling formulations 

•  Further justification, such as why bridging is appropriate (e.g. in-service monitoring)  

It is understood that extensive field data or bridging data may not be available when 
established biocides have been introduced into products based on new technology or new 
active substances are being developed. Field tests from different ships have limited value for 
the purpose of comparing efficacy due to the diversity of operational patterns and trading 

 
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routes and the likeliness for unforeseen circumstances or incidents not recorded. This, together 
with the complexity with respect to application and monitoring and the long exposure times 
required, explain why in-service tests are normally not available for new antifouling products. 
However, when data on in-service/field tests are available, these should be submitted as 
additional information. 

However, field data are required at renewal of a product authorisation, as the product will have 
been on the market for several years by this point. Further guidance on how to perform and 
assess these data will be developed in the future and incorporated into this guidance. 

5.7.1.1.8.4 Replication of efficacy tests 

Antifouling paints are normally tested in series during product development, where panels 
treated with a range of formulations, with only small variations between them, are tested to 
assess the effects of exposure on other paint properties, as well looking at the efficacy of the 
formulations. 

Since the testing takes place in a natural environment, the variation in fouling propagation and 
intensity between different years at the same test site will vary. A variable natural 
environment, the differences in fouling activity between years, and the criteria for establishing 
efficacy (the general nature of a label claim) make very detailed evaluations unnecessary.  

However, to increase the scientific rigour of the evaluation, the results of three replicate plates 
should be submitted. 

It is acknowledged that it is not common practice to test multiple replicates of individual 
formulations, however panels treated with similar formulations containing the same 
combination and concentration of active substances may be considered replicates when these 
are supported by a suitably robust reasoned case explaining the relevance of these 
formulations to the candidate product. The results from such panels should be submitted, along 
with details of the formulations used, as well as the reasoned case. 

5.7.1.1.9  Standard test methods 

5.7.1.1.9.1 Simulated use test methods 

The standard test methods available for the generation of simulated field data through raft 
testing of antifouling coatings are: 

1.  Efficacy evaluation of antifouling products. Conduct and reporting of antifouling 
efficacy evaluation trials. CEPE Antifouling Working Group, June 2012. This 
methodology has also been adopted by the International Paint and Printing Ink Council 
– IPPIC and presented at Technical Meeting I 2013 PT 21 efficacy workshop (Appendix 
21). 

2.  American Society of Testing Methods (ASTM) - ASTM D3623 - 78a (2004) Standard 

Test Method for Testing Antifouling Panels in Shallow Submergence which is linked to 
ASTM D6990-5(2011) Standard Practice for Evaluating Biofouling Resistance and 
Physical Performance of Marine Coating Systems. 

Reports based on both the above methods should be accepted. 

However, it should be noted that the ASTM methods were primarily developed to satisfy the 
detailed requirements of the US Navy and are not commonly used by the general antifouling 
industry. The main reasons for this are that they are resource intensive (in terms of the level of 
detail required in both the materials used as well as the analysis and reporting of the fouling 
species [including the number and diameter of individual organisms), thereby exceeding the 
requirements for substantiating a general product label claim (since normally specify only the 
general types of fouling and their extent are reported for regulatory purposes)] and that they 
specify relatively dated materials (paints), for which better and more applicable alternatives 
are available. Notwithstanding, the methods may provide a good basis for biological research. 

5.7.1.1.9.2 Field/In-service tests 

There are currently no national or international standards that cover field evaluation of 
antifouling products. Field tests (application on ships) are rarely used to screen formulations 
and establish the basis for an efficacy claim since they are time consuming and costly and since 
the results are heavily dependent upon the operations of individual vessels. To the extent field 

 
 
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trials are used, their purpose is normally to determine relative differences in efficacy between 
already commercial formulations during different use conditions (such as vessel speed, idle 
times, etc.). 

Typically a new antifouling paint represents an incremental improvement or an adaptation to a 
specific user requirement. Normally, therefore, the experience from similar commercial 
products will contribute to the confidence the manufacturer has with respect to the efficacy of a 
new product. 

However, at the point of renewal of a product authorisation, a product will have been on the 
market for several years and field data should be generated to demonstrate the actual 
performance of the product in use. 

5.7.1.1.10 Resistance 

Resistance is discussed in the general part of the TNsG on Product Evaluation in Chapter 6. A 
review of resistance is part of the evaluation at product authorisation. If new information is 
available which was not reviewed during the approval of active substance, this information 
should be provided at the time of product authorisation. 

In general development of resistance is not to be expected for marine use, as ships are treated 
with several antifouling paint products containing different active substances. However, this 
may not be the case for use in fresh water and aquaculture. 

5.7.1.1.11 Reports of development of resistance should always be mentioned. Service 
life 

Amateur antifouling products for recreational crafts are normally claimed to last for one 
yachting season, and are recommended to be retreated annually. Commercial vessels will have 
extensive tailor made paint specifications depending on their dry-docking interval and 
operational pattern. Different products and film thicknesses are frequently used at different 
parts of the vessel due to different light conditions and hydrodynamic forces. In the case where 
a label claim includes different types of use (e.g. both vessels and static installations), the 
corresponding protection times may differ. 

With respect to the ability of fouling organisms to settle and attach, static conditions are much 
more favourable than the conditions on vessels that are only idle for relatively short periods at 
the time. This together with the greatest levels of marine growth occurring in near shore 
conditions (as described in 2.1), explain why static raft testing is a worst case test. For 
recreational craft, however, the use conditions may be very different. Therefore, tests are 
frequently carried out for the same number of fouling seasons as the recommended use. 

It is not obligatory to state on the label what the service life of a product will be. 

5.7.1.2 Products intended for marine use 

5.7.1.2.1 Introduction 

Raft tests represent worst case conditions with respect to fouling intensity due to their static 
nature and because the tests are carried out in near shore environments. As the release of 
active substances from antifouling paints is assisted by hydrodynamic forces (i.e. through 
polishing), fouling will be more severe on static surfaces compared with moving boats and 
ships.  

Coastal waters are known to have the highest fouling intensity. The littoral zone along coasts 
constitutes a tiny part of the world’s oceans, but contributes markedly to the total marine 
production. The reason is that benthic production (per unit surface area) exceeds pelagic 
production by a factor of ten. Coastal macrophytes account for two-thirds of the total biomass 
of marine photo-synthetic organisms although they can only inhabit less than 0.5 % of the 
surface area of the oceans39. Therefore, when efficacy is demonstrated in coastal waters (the 
worst case situation), a product is also assumed to be effective in open sea and brackish 
conditions, and the data can be used to support these uses. 

39  R.S.K.  Barnes  and  R.N.  Hughes.  An  introduction  to  Marine  Ecology.  Blackwell  Scientific  Publications, 
1986. Page 37-39 

 
 
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5.7.1.2.2 Dossier requirements 

A report of the results from efficacy testing may also include the following about the test site, 
the test procedures, and the data reported: 

•  Method of application and information on the panel type and panel preparation; 

•  Location, geography, and water exchange conditions; 

•  Water temperature and salinity, including seasonal variations; 

•  Orientation, dimensions, and exposure depth of the test surface; 

•  Dimensions and type of material of test panels; 

•  Identity of the tested product and the control(s); 

•  Details on the panel preparation (application technique, possible primer paint, paint 

film thickness, number of coats); 

•  Date and duration of test; 

•  Date and raw data from each individual assessment of a test panel; 

•  Photos of test panel and control(s); 

•  The overall fouling assessment rating at each inspection during the exposure period; 

•  A description of the reporting company’s weighting system used to provide the overall 

fouling assessment rating. This should include how fouling coverage has been weighted 
in order to provide an overall efficacy assessment. The description should be 
transparent and explicitly explain the calculations carried out. (See example in 
Appendix 22); 

•  An interpretation of the data including a conclusion and a discussion of the validity of 
the results relative to the unprotected reference and the label claim for the product 
tested. 

5.7.1.2.2.1 Testing and field trials 

The recommended method for demonstrating efficacy of marine antifouling products is static 
raft testing. Raft testing allows a high number of formulations to be tested at worst case 
conditions. 

At least one raft test in European coastal waters should be provided. Test in Atlantic or 
Northern European Seas are preferred; however, other European waters are acceptable too. It 
is preferable to also provide the reports from additional tests, although these additional tests 
can be performed in other locations (e.g. in Europe or elsewhere in the world). At least three 
replicate panels should be provided per product (see section 5.7.1.1.8.4 for more information 
on replication of tests). Tests should be performed for at least one fouling season, which is at 
least six months covering the period of peak fouling activity. 

5.7.1.2.3 Assessment of authorisation 

The ability a product has to produce an antifouling effect is determined by a combination of the 
activity of the active substance(s) and the mechanical/physico-chemical properties of the paint. 
Parameters that will define the efficacy of an antifouling product include: 

•  The potency and release rate of the active substance(s) 

•  Operational patterns (e.g. speed, idle times, dry-docking interval, etc.) 

•  Physico-chemical conditions of the water and other climatic, seasonal, or local factors 
affecting fouling intensity (e.g. concentration of nutrients, hours of daylight, salinity, 
temperature, presence of ice, turbidity, etc.) 

The efficacy data submitted in support of an application represent part of the information 
assessed to establish if the product has the claimed level of efficacy. It is recognised that the 
actual in-service performance of an antifouling product will be dependent on a range of factors, 
which may include how and where a boat or vessel is operated, seasonal and annual variations, 
as well as the specifics of the antifouling coating itself. Commercial vessels receive tailor-made 
product specifications in order to meet various planned (and unforeseen) operational 
conditions. Thus, the general efficacy of a product under typical fouling conditions according to 
criteria in paragraph 2.3.1 should be demonstrated. 

5.7.1.2.3.1 Norms and criteria 

 
 
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The purpose of an efficacy test is to support the label claim. Efficacy is evaluated by comparing 
the extent of fouling on the test substrate with the fouling on a similar, but unprotected 
substrate which has been exposed simultaneously and at the same site. 

Fouling coverage is frequently evaluated based on the coverage of the typical marine fouling 
species such as slimes, algae and animals (barnacles, mussels, etc.). 

The three types of fouling species (slime, macro-algae and animals) may be rated differently 
when merged to an overall fouling assessment for the tested product since slime fouling is less 
significant compared to macro-fouling (for instance for the fuel consumption and 
manoeuvrability of a ship). An overall fouling assessment may describe the efficacy of a panel 
in categories such as for instance: 'Excellent', 'Good', 'Fair', and 'Poor'. An example to illustrate 
how the coverage of the main categories of fouling may be combined to provide an overall 
fouling assessment is given in Appendix 22. 

Since different companies may use different overall fouling assessment systems and 
interpretation of the result may vary with the type of product (what is 'poor' efficacy for marine 
water vessels might be 'good' for fresh water yachts), these ratings are not used as the 
pass/fail criterion for authorisation. Instead, the percentage fouling on the control and test 
panels is used. 

Normally, when tested in marine waters, the negative control will have at least 75 % fouling 
coverage at the end of the test. In this case, the result from a product under test should be 
acceptable if the coverage of macro-fouling on the panels is below 25 %. Macro-fouling is 
defined as large, distinct multicellular organisms visible to the human eye such as barnacles, 
tubeworms, or fronds of algae40. Algae shorter than 5 mm should be regarded as micro-fouling, 
together with slimes. 

If the 25 % criterion is not met, a justification should be provided for why the product may still 
be regarded as sufficiently efficacious for the intended use. 

5.7.1.3 Products for freshwater use 

5.7.1.3.1 Introduction 

Fresh and brackish waters are known to represent a less severe fouling challenge compared to 
marine waters. Effective antifouling protection may be environmentally important even where 
the general fouling challenge is low. For example, to reduce the risk of translocating invasive 
species (such as zebra mussels) into or between inland waterways, lakes, or brackish seas. 

5.7.1.3.2 Dossier requirements 

See 5.7.1.2.2 for the requirements on reporting the test procedure and data. 

5.7.1.3.2.1 Testing and field trials 

For products intended for use in both fresh water and marine waters, a raft test in marine 
coastal water is sufficient and a separate efficacy test under fresh water conditions is not 
normally carried out for. Since fresh and brackish waters are known to represent a less severe 
fouling challenge compared to marine waters, it is common practice to use the bridging 
principle and refer to tests conducted in marine waters. 

For products only intended to be used in fresh water, at least one raft test in fresh water 
should be provided. When raft tests are carried out in fresh water, the test site should be one 
known to have relatively high fouling levels, preferably in an area where zebra mussels are 
present. However, it is preferable to also provide the reports from additional tests. At least 
three replicate panels should be provided per product (see section 5.7.1.1.8.4 for more 
information on replication of tests). Tests should be performed for at least one fouling season, 
which is at least six months covering the period of peak fouling activity. 

5.7.1.3.3 Assessment of authorisation 

See section 5.7.1.2.3. 

40 IMO’s 2011 Guidelines for the Control and Management of Ship’s Biofouling to Minimize the Transfer of 
Invasive Aquatic Species, Section 2.1. Definitions. 

 
 
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5.7.1.3.3.1 Norms and criteria 

The purpose of an efficacy test is to support the label claim. Efficacy is evaluated by comparing 
the extent of fouling on the test substrate with the fouling on a similar, but unprotected 
substrate which has been exposed simultaneously and at the same site. 

In the case that an efficacy test is carried out in fresh water, it should be noted that as the 
fouling challenge is low, a 75 % or more coverage of fouling organisms on a negative control 
test panel cannot be expected. Therefore, if a test is carried out where micro-fouling is 
predominant and the coverage of macro-fouling is less than 75 %, the test may still be valid. 
In the case where less than 75 % of the surface of the negative control is covered with fouling, 
an explanation should be provided for why the test should be considered valid. 

It is also possible that in freshwater, macro-fouling (such as freshwater hydrozoans or zebra 
mussels) may completely cover a negative control. 

For tests in fresh water where the control panel has 75 % or more coverage of fouling 
organisms, the result from a product under test should be considered acceptable if the 
coverage of macro-fouling on the panels is below 25 %. 

For tests in marine water see Section 5.7.1.2.3 for criteria. 

5.7.1.4 Products for use in aquaculture 

5.7.1.4.1 Introduction 

In aquaculture use, antifouling products are used to treat infrastructure, including immersed 
structures such as cages, nets, ropes, buoys and pontoons, as well as equipment such as 
pipelines, pumps, filters, and holding tanks. 

5.7.1.4.2 Dossier requirements 

See 5.7.1.2.2 for the requirements on reporting the test procedure and data.  

5.7.1.4.2.1 Testing and field trials 

Relevant field or simulated use trials should be provided to demonstrate the efficacy under in-
use conditions. Static testing closely resembles real life conditions for aquaculture use. Test 
surfaces may include panels and net/cage samples suspended securely from the raft. 

At least one field test should be provided. However, it is preferable to also provide the reports 
from additional tests. At least three replicates should be provided per product (see section 
5.7.1.1.8.4 for more information of replication of tests). Tests should be performed for at least 
one fouling season, which is at least six months covering the period of peak fouling activity. 

5.7.1.4.3 Assessment of authorisation 

The ability a product has to produce an antifouling effect is governed by mechanical and 
physico-chemical properties of the paint. Relevant parameters to be taken into account when 
assessing the efficacy of an antifouling product include: 

•  The potency and release rate of the active substance(s) in the paint 

•  Physico-chemical conditions of the water and other climatic, seasonal or local factors 
affecting fouling intensity (e.g. concentration of nutrients, hours of daylight, salinity, 
temperature, presence of ice, turbidity, etc.) 

A report of results from efficacy testing should include the following information about the test 
site, the test procedures, and the data reported: 

•  Method of application (e.g. dipping of nets) and type of test substrate 

•  Location, geography, and water exchange conditions 

•  Water temperature and salinity 

•  Orientation, dimensions, exposure depth of test surface, and date and duration of the 

test 

•  The extent and main categories of fouling and an interpretation of this relative to an 

unprotected surface and the label claim for the product tested 

5.7.1.4.3.1 Norms and criteria 

The purpose of an efficacy test is to defend the label claim. Efficacy is evaluated by comparing 
the extent of fouling on the test substrate (panel, cage, net, etc.) with the fouling on a similar, 

 
 
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but unprotected substrate which has been exposed simultaneously and at the same site. 
Efficacy is demonstrated if fouling on the treated surface is considerably reduced compared to 
the fouling on the unprotected surface. 

Fouling coverage is frequently evaluated based on the coverage of typical fouling species. 
These ratings are then merged to provide a consolidated figure for the three major types of 
fouling species: slime, macro-algae and animals (see Appendix 22). The three types may be 
rated differently when combined to an overall fouling assessment for the tested product. For 
example, slime fouling is less significant compared to macro-algae and large hard animals for 
the water exchange through nets and cages. 

If a product for aquaculture use is tested on panels, the pass/fail criteria for the test may be 
the same as in paragraph 5.7.1.2.3. 

5.7.2 PT22 Embalming and taxidermist fluids 

5.7.2.1 General introduction 

Annex V of BPR defines Product Type 22 products as follows: "Embalming and taxidermist 
fluids. Products used for the disinfection and preservation of human or animal corpses, or parts 
thereof". Embalming for this purpose only aims at the temporary preservation of the deceased 
person, before burial. Taxidermy fluids and those intended for long-term preservation (e.g. 
repatriation as shipping cases) are not covered by this guidance document. These particular 
cases will be taken into account in a future update and inclusion into Volume II Part B of the 
new BPR guidance structure. 

This guidance document is intended for applicants to assist them in compiling an authorisation 
request dossier regarding the efficacy aspect, and thus specifies the general conditions for 
carrying out efficacy assessments of biocidal products for marketing authorisations. 

This guidance document may be reviewed in the event of regulatory changes or technical 
advances. 
5.7.2.2 Use of the products 

5.7.2.2.1 The issue of bodily decomposition 

5.7.2.2.1.1 Physical, chemical and microbiological post-mortem activities 

A body starts to decompose as soon as the blood ceases to circulate and oxygen is no longer 
supplied to the tissues. Under conditions favourable to decay, the body cools in the first few 
hours after death, dehydration sets in (lividity) together with rigor mortis resulting from 
anaerobic hydrolysis of muscle glycogen. The first stages of cell degradation can be seen with 
the onset of lividity. 

The natural degradation of the body's organic matter results from the action of enzyme, tissue 
and microbial processes. The ecosystem whose characteristics determine the succession of 
physical, chemical and microbiological changes that occur post mortem can be defined as the 
set of interactions between ambient factors (temperature, hygrometry), individual factors, 
especially the body's water, muscle and fat composition, and the body's own microbial flora, 
both external (skin) and internal (digestive and respiratory). Together, these conditions affect 
the establishment, acclimatisation and development of the dominant indigenous flora, 
separately or in association, and thus steer the metabolism towards either speed or slow 
decomposition. 

The activity of the microflora, initially latent, intensifies; the first stages of mineralisation of the 
organic matter, stages of the nitrogen, carbon, oxygen and hydrogen cycles, constitute both 
superficial and profound decomposition. This decay is defined partly by the decomposition of 
the organic tissues, mainly under the influence of the bacteria hosted by the individual, 
especially those in the intestinal flora, and then by fungi, and partly by the decomposition of 
the organic matter and the bacteria responsible for mineralisation that gradually invade the 
body, via the body fluids. 

As the proteins, lipids and certain carbohydrates that provide the substrate degrade; they 
produce malodorous soluble and gaseous substances, containing sulphur, nitrogen and 
carboxylates. Depending on the specific activities developed by the flora in place, the resulting 

 
 
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foul odours can vary in nature and intensity. It is increased by higher temperatures and by 
interferences between chemical groups. As degradation progresses, the source of foul odours 
moves gradually from the body itself to the liquid products of decay, which rapidly become the 
principal source of foul odours. As the organic matter becomes hydrolysed into more soluble 
compounds it becomes easier for microorganisms to assimilate them, facilitating the production 
of foul odours. 

5.7.2.2.1.2 The micro-organisms involved 

• 

in the early stages of decomposition of the liquids and soft tissues (with production of 
gases), only the following species are found: Pseudomonas fluorescens and Micrococcus 
ureae; 

•  at a later stage of lipid transformation, the following appear: Pseudomonas sp. and then 

Pyogenes sp. 

The initial wave consists of aerobic bacteria while those following are anaerobic (Diplococcus 
magnus, Streptococcus sp., Serratia liquefaciens, Bacteriodes sp. etc.). This decomposition of 
the body due to bacteria and saprotrophic fundi gradually leads to autolysis of the remains, 
which is pursued later and over time by the bacteria active in the mineralisation of the organic 
matter, although this last stage is related to the level of humidity. Various factors concerning 
the environment of the body intervene (humidity, temperature, aeration) as well as its size, 
age, causes of death and place of storage. 

The decay is predominantly influenced by the bacteria that had been hosted by the individual, 
especially those in the intestinal flora. The bacterial species frequently found in decomposing 
bodies are: 

•  of intestinal origin: enterobacteria, especially Escherichia coli; clostridia, especially 

Clostridium tetani, C. welchii and C. difficile; and faecal Streptococcus; 

•  of dermal origin: Staphylococcus spp.; 

•  of environmental origin: Bacillus spp. 

The saprotrophic fungi and yeasts succeed one another in specific groups and the flora changes 
in line with the gradual alteration of the substrate, which thus provides a choice habitat for 
certain species of mycota at one moment and not at others. 

The decomposition of the body due to bacteria and saprotrophic mycota accelerates the 
alteration started by autolysis, before the mineralising bacteria that invade the body later bring 
it into the cycle of waste material in the biosphere. 

There may also be other pathogenic micro-organisms, such as the tuberculosis bacillus 
(Mycobacterium tuberculosis) or other mycobacteria, or again viruses such as hepatitis or 
Human Immunodeficiency Virus (HIV), which can persist in the body. 

5.7.2.2.2 Products for preserving human bodies and their uses 

5.7.2.2.2.1 Types of application  

The embalmer begins by physically working the limbs to reduce lividity and facilitate the flow of 
the preserving fluid. This is used for two separate purposes and at different concentrations: 

•  arterial fluid: an aquaeous solution injected under pressure into the vascular system 

(the embalmer adjusts the final concentration to the condition of the body). This liquid 
is injected in the arterial system via the carotid or the femoral artery (sometimes at 
several points if diffusion is poor). The injection is made under pressure (by pump) or 
by gravity. This result in venous drainage: replaced by the injected product, the blood 
leaves the body via the jugular vein. Six to ten litres are injected and four litres (of 
blood and other body fluids) are removed by suction; 

• 

cavity fluids: these are usually used at high concentration to preserve the thoracic and 
abdominal cavities, which cannot be irrigated by arterial injection. Using a trocar 
connected to a pump, about two litres of the pure undiluted solution are injected into 
the peritoneal cavity through an incision close to the navel. 

There are also preparations for dermal use. These are gels designed to limit the decomposition 
of the body by treating bedsores. For this type of product, applicants must complete the 
appropriate section of the assessment grid, demonstrating the efficacy of the product. 

 
 
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In addition to its biocidal active substance(s), such a formulation could include the following co-
formulants, which must have no biocidal activity: 

•  anticoagulants: to fluidify the product and ensure correct diffusion (sodium chloride and 

sodium citrate); 

•  hydrating and moistening agents: to slow the drying out of the body by hydrating the 
tissues and making them more supple (glycerine, ethylene glycol, propylene glycol, 
hexylene glycol, urea); 

• 

• 

surface-active agents: to facilitate adsorption of the fluid and penetration of the 
membranes and to maintain the solubility of the other components of the formulation, 
which are generally cations, as these surfactants are often also antimicrobials; 

colouring agents: to ensure that the fluid is of a colour similar to blood; synthetic 
colouring agents are generally used (eosin, erythrosine or food colouring agents); 

•  perfumes. 

5.7.2.2.2.2 Products used for aesthetic purposes 

Preservation may be supplemented with aesthetic treatment involving remodelling the face 
(modelling wax), sewing or bonding together the upper and lower jaws, placing eye caps under 
the eyelids to keep the eyes closed (or possibly gluing them shut). Finally, when all other 
treatment has been completed, cosmetic make-up may be applied, partly to give a more 
agreeable appearance but also partly to delay dehydration. 

These products are not considered during assessment of the efficacy of the preservation 
product. However, if these products contain substantial amount of active substance and claim 
an effect on bodily composition, they should be considered as biocide. 

5.7.2.3 Data required 

5.7.2.3.1 Claims and labelling 

When an application for the approval of a PT 22 substance is being assessed, the evaluation of 
the efficacy is focused on the efficacy of the biocidal product and not on the other products (as 
cosmetic) which can be also included in an embalming treatment, so this aspect must be 
demonstrated unambiguously in laboratory tests and tests on human bodies, the details of 
which must be available on request. 

As a minimum, a PT 22 product must claim to be active against a broad spectrum of bacteria; 
yeasts, fungi and viruses are considered as an additional spectrum. As explained above, 
bacteria are the principal micro-organisms targeted by PT 22 products. Yeast, fungi and viruses 
have less relevance in the early stages of bodily decomposition. 

Nonetheless, an active substance with a broad spectrum on different types of micro-organism 
would provide better protection for users (e.g. against tuberculosis bacilli, hepatitis viruses or 
HIV, etc.). 

5.7.2.3.2 Efficacy tests 

5.7.2.3.2.1 Laboratory tests 

As there is currently no standardised method recognised at European level targeting the scope 
covered by PT 22 products, and as no technical reference documents were found either in 
France or throughout the world, it is important that methods used should achieve two different 
yet complementary goals: 

• 

• 

the rapid destruction of bacteria, representative of the bacterial sphere, in the presence 
of a strongly interfering organic load simulating the bodily fluids; 

to maintain this antibacterial activity for several days, thus demonstrating that there is 
no subsequent proliferation of these micro-organisms. 

5.7.2.3.2.2 Determining bactericidal activity  

As already mentioned above, the minimum claim is a bactericidal activity. Other additional 
activities, such as fungicide or virucide activities must be supported by relevant tests. 

From among the techniques available, the selection was made based on the following criteria: 

 
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•  a method that has been standardised at least at European level – the bacterial 

“suspension” test used in the medical sector  

• 

the presence of a standardised strong organic load accurately simulating organic bodily 
fluids. 

In compliance with the classification of European standards (EN 14885), the two tests selected 
belong to the categories of tests in Phase 2, Step 1 which include quantitative suspension tests 
for establishing that a biocidal product has a bactericidal activity by simulating its use under 
real conditions: 

a)  tests according to the EN 13727 standard: this mandatory test determines the minimum 
bactericidal concentration of a product on the basis of a 5-log reduction in titre of a 
bacterial suspension, at a temperature of 20°C, for 60 minutes of contact, in the 
presence of a strong organic load (bovine albumin 3 g/L + ovine erythrocytes 3 ml/L), 
on three species of bacteria (Staphylococcus aureus ATCC 6538, Pseudomonas 
aeruginosa ATCC 15442, Enterococcus hirae ATCC 10541); 

b)  tests according to the EN 14348 standard: this additional test must be taken into 
account if the applicant advances any claim concerning activity against agents 
responsible for tuberculosis, or if complementary tests prove necessary to cover this 
particular need. This test has a methodology similar to that for the previous test, 
determining the minimum tuberculocidal concentration of a product on the basis of a 4-
log reduction in titre of a bacterial suspension, at a temperature of 20°C, for 60 minutes 
of contact, in the presence of a strong organic load (bovine albumin 3 g/L + ovine 
erythrocytes 3 ml/L), on the bacterium Mycobacterium terrae ATCC 15755. 

Any claim by applicants that a product targets a specific micro-organism must be supported by 
supplementary studies. For example, a claim of activity against the agents responsible for 
tuberculosis must be verified in compliance with the EN 14348 standard. If there is no 
recognised standard for a specific micro-organism, the EN 14348 standard may be used for the 
micro-organism in question. 

The most recent version of standards in force at the time of the tests must be used. 

Furthermore, in accordance with the conclusions in Annex VI (77) the level, consistency and 
duration of protection, control or other intended effects must, as a minimum, be similar to 
those resulting from suitable reference products, where such products exist, or to other means 
of control. Where no reference products exist, the biocidal product must give a defined level of 
protection or control in the areas of proposed use. 

Considering the history of the use of formaldehyde, it may therefore be worthwhile to include 
with the application information about the bactericidal efficacy of formaldehyde, if available. 

In France, formaldehyde is most commonly used at concentrations of about 28% for cavity 
fluid and 1.5% for arterial fluid. As formaldehyde is currently under assessment in the review 
programme, efficacy data may become available when the assessment report is published by 
the evaluating Competent Authority (eCA). The standards proposed above for validating claims 
may be reviewed at a later stage in the context of the review of this guidance document as a 
result of the conclusions published by the eCA on the efficacy of formaldehyde, or in the event 
of other data for this same substance becoming available in the future. 

5.7.2.3.2.3 Verifying that antibacterial activity is maintained 

When embalming, the biocidal product must remain effective over several days, until burial. 
The persistence indicated on the label must be proven, e.g. by challenge tests. The following 
protocol may be used, adapted from the French NF X30-503 standard (Healthcare waste - 
Reduction by disinfection pre-treatment appliances in microbiological and mechanical risks 
involving infections and other comparable healthcare waste). 

• 

In order to ensure that bacteria are destroyed and not merely subjected to stress or 
inhibition by the biocidal product, and to confirm the absence of bacterial revival, the 
bacterial suspension, treated according to the EN 13727 standard, is held at ambient 
temperature for four to six days and then the bacteria are counted. In the laboratory, it 
is held at 20°C until analysis. 

•  The bacteria in the bacterial suspension are counted on the day of treatment and again 

after four to six days. 

 
 
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•  Lasting disinfection is shown by the absence of bacterial revival, i.e. the bacterial count 
on day 4-6 must not be increased by more than one log compared to the bacterial load 
measured in the sample taken on the day of treatment (Day 0). 

•  The "effective" dose of the product must be in a range bounded by upper and lower 

limits, which are: 

o 

a lower concentration for which bacterial recrudescence is observed after 4-6 
days; 

o  a higher concentration. 

5.7.2.3.2.4 Tests on human bodies  

To complement in vitro efficacy tests for the biocidal product used for the preservation of 
human bodies, tests on bodies are necessary to assess product performance. 

Because of the number of factors that can influence the efficacy of a biocidal product, such as 
the cause of death or the time lapsed or the condition of the body before embalming begins, a 
sufficient number of bodies (at least 20) satisfying the requirements of the grid in Appendix 23 
and the claims for the product, must be available for optimum assessment of the results in 
terms of preservation of the body for viewing by families. 

NOTE to the reader: 
The applicant has to inquire about the legislation in force in the Member State (MS) 
where the tests on human bodies are performed (e.g. current French regulations 
only allow bodies donated to science to be used to test a product that has not yet 
been approved. 

Every centre for the donation of bodies participating in these tests on human bodies must 
declare the number of bodies undergoing tests in its establishment. This declaration is supplied 
to the applicant and must be submitted with the application). 

In all cases, whatever the legislation in force in each MS, tests on human bodies with good 
quality and in line with this guidance will be accepted by MS when the dossier will be submitted 
for authorisation. 

The assessment grid for specific biocidal products is shown in Appendix 24. Its purpose is not 
to assess the overall embalming treatment but only the biocidal product for which authorisation 
is being requested. 

The grid consists of: 

•  general information: date and place of the treatment, identification of the deceased 

(gender, age), weight, corpulence, adiposity, date and causes of death, etc.; 

• 

• 

the preoperative body examination: bodily integrity, autopsy, external prostheses, 
surgery, visible anomalies (decomposition, rigidity, dehydration, lividity, colouring of 
tissues, dermal lesions, distension of the abdomen, bruising, etc.). The bodies used 
must be representative of the range of criteria listed in this section; 

the techniques used to inject the biocidal product: timetable, sites and types of 
injection, biocidal product used, drainage and puncture; 

•  observations concerning the injection of the biocidal product: observations during 

treatment, 48 hours after treatment and after different periods in accordance with the 
applicant's claims; 

•  where necessary, the use of other products during the preservation process: products 

for cosmetic purposes, humidifiers and other products. 

The embalmer thus assesses the efficacy of the embalming product on a series of human 
bodies, using the grid provided. The efficacy is judged for the duration claimed by the 
manufacturer according to observations concerning odour, colouring and the suppleness of the 
skin after injection of the biocidal product. In the event that the tests on these human bodies 
have to be interrupted for any reason, the results already obtained remain valid for three years 
following the official decision to halt the tests. 

5.7.2.3.2.5 Choice of dose  

 
 
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The usage dose41 claimed is a matter for the applicant. Indeed, related to the body conditions, 
it can be necessary to test several doses above the dose determined in laboratory and then 
define a range of doses, adapted to difficult cases. They must choose the usage dose claimed 
according to the efficacy sought and the precautions for use that will be imposed on embalming 
technicians by their employers, depending on the health risks created by the full preparation 
(active substance at the chosen concentration plus excipients and solvents). In cases where 
little is known about the pathogenic micro-organisms that might present a risk to the 
embalmer, it is essential that protective measures be taken during the preservation process. 
These measures should not be primary criteria for choosing the biocidal product used for the 
treatment. 

If the applicant chooses a range of doses instead of a single value, the lower must be justified 
with appropriate tests, as defined in the preceding section (and also the higher dose in the case 
where different doses have been tested in the human body tests to cover difficult cases). The 
applicant may also request approval for two different doses, one of them more concentrated for 
special or difficult cases (bodies found some time after death or in contact with water, for 
example). 

5.7.2.4 Assessing the application for authorisation 

The assessment of the embalming product shall be favourable if it satisfies the following 
efficacy criteria: 

• 

• 

• 

laboratory test: bactericidal properties (EN 13727 and/or EN 14348 standards): 
obligatory test conditions; 

laboratory test: e.g. challenge test: no bacterial recrudescence for at least 4-6 days by 
more one log compared to the bacterial load measured in the sample taken on the day 
of treatment (Day 0), with the bacterial suspension being held at ambient temperature; 

field test: 80% of the bodies must meet the satisfaction criteria at T+48 hours. 
Satisfaction criteria are according to the grid: normal or fair odour, colouring and 
suppleness of the skin, related to the initial conditions of the body. 

SUMMARY OF THE PARAMETERS ASSESSED 

EFFICACY CLAIMS ON THE LABEL SUBMITTED 

1. Does the applicant make any specific claims? Y/N 

2. Have the efficacy claims on the label been judged and dealt with according to the 
parameters described in this guidance document for this type of product? Y/N 

ASSESSING THE DATA 

3. Has each study (or supplementary item) been assessed individually for robustness? Y/N 

4. Has each study (or supplementary item) been assessed individually for quality 
assurance? Y/N 

5. Has each study (or supplementary item) been assessed individually for suitability (i.e. 
for reliability and relevance concerning the claims)? Y/N 

DECISION-MAKING 

Considering all the available data: 

6. Are the claims on the label sufficiently supported? Y/N 

7. Do the claims on the label require modifications? Y/N 

8. On the basis of the efficacy data submitted, can authorisation for the use of the product 
be recommended? Y/N 

41 Concentration and volume injected 

 
 
 
 
 
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Appendix 1. Claims Matrices 

The claims matrices are a set of tables linked to this guidance document: these documents are 
available on the ECHA Biocides Efficacy Working Group webpage [http://echa.europa.eu/about-
us/who-we-are/biocidal-products-committee/working-groups/efficacy.]. 

The claims matrices linked to this document are intended to cover biocidal products covered 
under the scope of Product Type 1, 2, 3 and 4 and for Treated Articles. 

The claims matrix is a tool for the applicant and CAs. It is intended to capture the information 
that  is  needed  in  the  authorisation  dossier,  to  adequately  describe  typical  combinations  of 
products,  formats  of  application  of  the  products,  as  well  as  target  sites.  It  also  includes  the 
claims  made  and  the  requirements  for  testing  these  claims  (in  terms  of  methodology  and 
appropriate performance standards) for a product to be used in this way. 

The reader should note that the matrices are not exhaustive in terms of use patterns, scenarios 
and test methods. 

The  claims  matrix  must  be  used  together  with  the  relevant  sections  within  the  efficacy 
guidance document so as to provide both applicants and CAs alike with clear direction as to the 
nature and extent of the efficacy data required to support a claimed effect. The claims matrix 
acts as a guide to the information required when compiling an efficacy dataset for a PT1, PT2, 
PT3 or PT4 biocidal product and for Treated Articles. 

To note: 

•  Each row (entry) within the matrices is not independent and can be linked to other 

entries. 

•  These matrices only address biocidal claims made for these products. 

•  The claim matrix will be updated regularly according to the state-of-the-art. 

 
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Appendix 2. Standards  and  testing  methods  for  efficacy-
testing of disinfectant biocidal products (PT 1-5) 

The methods for testing efficacy referenced within this guidance document are enlisted below. 
The use of European Standards (Table 31) is highly recommended if available and appropriate 
for  the  respective  application42.  Should  no  European  Standard  for  an  application  be  available 
yet and an adaption of an existing standard is not possible according to the rules laid down in 
EN  14885,  other  test  methods  and  guidance  documents  (Table  32)  may  be  used.  In  cases 
where the below mentioned methods are inappropriate to demonstrate efficacy of a product for 
special  applications,  methods  from  other  national  or  international  standardisation  bodies  may 
also  be  employed.  These  include  for  example,  OECD,  ASTM  or  ISO  methods.  It  is 
recommended  to  agree  such  testing  strategies  with  the  evaluating  CA  before  tests  are 
performed. 

Tests  should  be  carried  out  according  to  the  respective  latest  edition  of  a  standard.  Please 
check the respective web sites for the latest information. 

Table 31: CEN European standards 

Reference 

Title 

PT 

Scope/Remarks 

EN 1276 

EN 1499 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
bactericidal activity of chemical 
disinfectants and antiseptics used in 
food, industrial, domestic, and 
institutional areas - Test method 
and requirements (phase 2, step 1) 

Chemical disinfectants and 
antiseptics - Hygienic handwash - 
Test method and requirements 
(phase 2, step 2) 

EN 1500 

Chemical disinfectants and 
antiseptics - Hygienic handrub - 
Test method and requirements 
(phase 2, step 2) 

EN 1650 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
fungicidal or yeasticidal activity of 
chemical disinfectants and 
antiseptics used in food, industrial, 
domestic, and institutional areas - 
Test method and requirements 
(phase 2, step 1) 

1,2,4  This European Standard specifies a method 
for testing bactericidal activity by assessing 
reduction in the number of viable bacterial 
cells in suspension under defined 
conditions. The approach can be applied to 
formulated products or to biocidal active 
substances. 

1 

1 

This European Standard specifies a test 
method simulating practical conditions for 
establishing whether a hygienic handwash 
product reduces the transmission of 
transiently contaminating micro-organisms 
when used to wash the artificially 
contaminated hands of volunteers. 

This European Standard specifies a test 
method simulating practical conditions for 
establishing whether a hygienic handrub 
product reduces the transmission of 
transiently contaminating micro-organisms 
when rubbed onto the artificially 
contaminated hands of volunteers. 

1,2,4  This European Standard specifies a method 

for testing fungicidal or yeasticidal activity 
by assessing reduction in the number of 
viable mould spores and/or yeast cells in 
suspension under defined conditions. The 
approach can be applied to formulated 
products or to biocidal active substances. 

42 The CEN does not sell or distribute standards or any other deliverable. All European Standards (EN) and 
drafts (prEN) as well as other approved documents are directly available for purchase from the CEN 
national standardisation bodies. 

 
 
 
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Reference 

Title 

PT 

Scope/Remarks 

EN 1656 

EN 1657 

EN 12353 

EN 12791 

EN 13610 

EN 13623 

EN 13624 

3 

3 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
bactericidal activity of chemical 
disinfectants and antiseptics used in 
the veterinary area - Test method 
and requirements (phase 2, step 1) 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
fungicidal or yeasticidal activity of 
chemical disinfectants and 
antiseptics used in the veterinary 
area – Test method and 
requirements (phase 2, step 1) 

1,2,3,
4,5 

1 

4 

Chemical disinfectants and 
antiseptics - Preservation of test 
organisms used for the 
determination of bactericidal 
(including Legionella), 
mycobactericidal, sporicidal, 
fungicidal and virucidal (including 
bacteriophages) activity 

Chemical disinfectants and 
antiseptics - Surgical hand 
disinfection - Test method and 
requirements (phase 2, step 2) 

Chemical disinfectants - 
Quantitative suspension test for the 
evaluation of virucidal activity 
against bacteriophages of chemical 
disinfectants used in food and 
industrial areas - Test method and 
requirements (phase 2, step 1) 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
bactericidal activity against 
Legionella of chemical disinfectants 
for aqueous systems - Test method 
and requirements (phase 2, step 1) 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
fungicidal and yeasticidal activity in 
the medical area - Test method and 
requirements (phase 2, step 1) 

1,2 

This European Standard specifies a method 
for testing bactericidal activity by assessing 
reduction in the number of viable bacterial 
cells in suspension under defined 
conditions. The approach can be applied to 
formulated products or to biocidal active 
substances. 

This European Standard specifies a method 
for testing fungicidal or yeasticidal activity 
by assessing reduction in the number of 
viable mould spores and/or yeast cells in 
suspension under defined conditions. The 
approach can be applied to formulated 
products or to biocidal active substances. 

This method specifies how to keep test 
organisms used and defined in European 
Standards for the determination of 
bactericidal, mycobactericidal, sporicidal, 
fungicidal and virucidal (incl. 
bacteriophages) activity of chemical 
disinfectants and antiseptics drawn up by 
CEN/TC 216. 

This European Standard specifies a test 
method simulating practical conditions for 
establishing whether a product for surgical 
hand disinfection reduces the transmission 
of the microbial flora on hands when used 
for the treatment of clean hands of 
volunteers. 

This European Standard specifies a method 
for testing virucidal activity against 
bacteriophages by assessing reduction in 
the number of infectious bacteriophage 
particles in suspension under defined 
conditions. The approach can be applied to 
formulated products or to biocidal active 
substances. 

for testing bactericidal activity against 
Legionella by assessing reduction in the 
number of viable Legionella cells in 
suspension under defined conditions. The 
approach can be applied to formulated 
products or to biocidal active substances. 

This European Standard specifies a method 
for testing fungicidal or yeasticidal activity 
by assessing reduction in the number of 
viable mould spores and/or yeast cells in 
suspension under defined conditions. The 
approach can be applied to formulated 
products or to biocidal active substances. 

2,4,5  This European Standard specifies a method 

 
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Reference 

Title 

PT 

Scope/Remarks 

EN 13697 

EN 
1370443 

EN 13727 

EN 14204 

Chemical disinfectants and 
antiseptics - Quantitative non-
porous surface test for the 
evaluation of bactericidal and/or 
fungicidal activity of chemical 
disinfectants used in food, 
industrial, domestic and institutional 
areas - Test method and 
requirements without mechanical 
action (phase 2, step2) 

Chemical disinfectants - 
Quantitative suspension test for the 
evaluation of sporicidal activity of 
chemical disinfectants used in food, 
industrial, domestic and institutional 
areas - Test method and 
requirements (phase 2, step 1) 

2,4 

4 
(1,2, 
3) 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
bactericidal activity in the medical 
area - Test method and 
requirements (phase 2, step 1) 

1,2 

3 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
mycobactericidal activity of chemical 
disinfectants and antiseptics used in 
the veterinary area - Test method 
and requirements (phase 2, step 1) 

EN 14347 

Chemical disinfectants and 
antiseptics - Basic sporicidal 
activity - Test method and 
requirements (phase 1) 

1,2,3,
4 

EN 14348 

1,2 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
mycobactericidal activity of chemical 
disinfectants in the medical area 
including instrument disinfectants - 
Test methods and requirements 
(phase 2, step 1) 

This European Standard specifies a method 
for testing bactericidal and/or fungicidal or 
yeasticidal activity by assessing reduction in 
the number of viable bacterial cells and/or 
mould spores and/or yeast cells dried on a 
steel carrier under defined conditions. The 
approach can be applied to formulated 
products or to biocidal active substances. 

This European Standard specifies a method 
for testing sporicidal activity by assessing 
reduction in the number of viable bacterial 
endospores in suspension under defined 
conditions. The approach can be applied to 
formulated products or to biocidal active 
substances. 

This European Standard specifies a method 
for testing bactericidal activity by assessing 
reduction in the number of viable bacterial 
cells in suspension under defined 
conditions. The approach can be applied to 
formulated products or to biocidal active 
substances. 

This European Standard specifies a method 
for testing mycobactericidal activity by 
assessing reduction in the number of viable 
mycobacterial cells in suspension under 
defined conditions. The approach can be 
applied to formulated products or to biocidal 
active substances. 

This European Standard specifies a method 
for testing sporicidal activity by assessing 
reduction in the number of viable bacterial 
endospores in suspension under defined 
conditions. The method is declared as a 
phase 1 test but, but based on its 
requirements, it can serve as a suspension 
test (comparable to phase 2, step 1) until 
revised/additional CEN methodology for 
testing sporicidal activity becomes available. 
The approach can be applied to formulated 
products or to biocidal active substances. 

This European Standard specifies a method 
for testing mycobactericidal activity by 
assessing reduction in the number of viable 
mycobacterial cells in suspension under 
defined conditions. The method is also 
applicable to demonstrate tuberculocidal 
activity only. The approach can be applied 
to formulated products or to biocidal active 
substances. 

43  EN  13704  is  under  review  and  the  revised  standard  will  include  veterinary  and  human  health  care 
areas. 

 
 
 
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Reference 

Title 

PT 

Scope/Remarks 

EN 14349 

EN 14476 

EN 14561 

EN 14562 

EN 14563 

EN 14675 

3 

Chemical disinfectants and 
antiseptics - Quantitative surface 
test for the evaluation of 
bactericidal activity of chemical 
disinfectants and antiseptics used in 
the veterinary area on non-porous 
surfaces without mechanical action - 
Test method and requirements 
(phase 2, step 2) 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
virucidal activity in the medical area 
- Test method and requirements 
(phase 2, step 1) 

1,2 
(4) 

2 

2 

2 

3 

Chemical disinfectants and 
antiseptics - Quantitative carrier 
test for the evaluation of 
bactericidal activity for instruments 
used in the medical area - Test 
method and requirements (phase 2, 
step 2) 

Chemical disinfectants and 
antiseptics - Quantitative carrier 
test for the evaluation of fungicidal 
or yeasticidal activity for 
instruments used in the medical 
area - Test method and 
requirements (phase 2, step 2) 

Chemical disinfectants and 
antiseptics - Quantitative carrier 
test for the evaluation of 
mycobactericidal or tuberculocidal 
activity of chemical disinfectants 
used for instruments in the medical 
area - Test method and 
requirements (phase 2, step 2) 

Chemical disinfectants and 
antiseptics - Quantitative 
suspension test for the evaluation of 
virucidal activity of chemical 
disinfectants and antiseptics used in 
the veterinary area - Test method 
and requirements (phase 2, step 1) 

EN 14885 

Chemical disinfectants and 
antiseptics - Application of European 
Standards for chemical disinfectants 
and antiseptics 

1,2,3,
4,5 

This European Standard specifies a method 
for testing bactericidal activity by assessing 
reduction in the number of viable bacterial 
cells dried on a steel carrier under defined 
conditions. The approach can be applied to 
formulated products or to biocidal active 
substances. 

This European Standard specifies a method 
for testing virucidal activity by assessing 
reduction in the number of infectious virus 
particles in suspension under defined 
conditions. The approach can be applied to 
formulated products or to biocidal active 
substances. 

This European Standard specifies a method 
for testing bactericidal activity by assessing 
reduction in the number of viable bacterial 
cells dried on a frosted glass carrier under 
defined conditions. The approach can be 
applied to formulated products or to biocidal 
active substances. 

This European Standard specifies a method 
for testing fungicidal or yeasticidal activity 
by assessing reduction in the number of 
viable mould spores and/or yeast cells dried 
on a frosted glass carrier under defined 
conditions. The approach can be applied to 
formulated products or to biocidal active 
substances. 

This European Standard specifies a method 
for testing mycobactericidal activity by 
assessing reduction in the number of viable 
mycobacterial cells dried on a frosted glass 
carrier under defined conditions. The 
method is also applicable to demonstrate 
tuberculocidal activity only. The approach 
can be applied to formulated products or to 
biocidal active substances. 

This European Standard specifies a method 
for testing virucidal activity by assessing 
reduction in the number of infectious virus 
particles in suspension under defined 
conditions. The approach can be applied to 
formulated products or to biocidal active 
substances. 

This European Standard specifies the 
European Standards, i.e. test methods, to 
which products have to conform in order to 
support the claims for microbicidal activity 
which are referred to in this document. It 
also specifies terms and definitions which 
are used in European Standards. It is 
applicable to products for which activity is 
claimed against the following micro-
organisms: vegetative bacteria (incl. 
mycobacteria and Legionella), bacterial 
spores, yeasts, fungal spores and viruses 
(incl. bacteriophages). 

 
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Reference 

Title 

PT 

Scope/Remarks 

EN 16437 

EN 16438 

EN 16615 

3 

3 

Chemical disinfectants and 
antiseptics - Quantitative surface 
test for the evaluation of 
bactericidal activity of chemical 
disinfectants and antiseptics used in 
veterinary area on porous surfaces 
without mechanical action - Test 
method and requirements (phase 2, 
step 2) 

Chemical disinfectants and 
antiseptics - Quantitative surface 
test for the evaluation of fungicidal 
or yeasticidal activity of chemical 
disinfectants and antiseptics used in 
the veterinary area on non-porous 
surfaces without mechanical action - 
Test method and requirements 
(phase 2, step 2) 

2 
(4) 

Chemical disinfectants and 
antiseptics - Quantitative test 
method for the evaluation of 
bactericidal and yeasticidal activity 
on non-porous surfaces with 
mechanical action employing wipes 
in the medical area (4-field test) - 
Test method and requirements 
(phase 2, step 2) 

EN 16616 

Chemical disinfectants and 
antiseptics - Chemical-thermal 
textile disinfection - Test method 
and requirements (phase 2, step 2) 

2 
(3, 4) 

EN 16777 

2 
(4) 

Chemical disinfectants and 
antiseptics - Quantitative non-
porous surface test without 
mechanical action for the evaluation 
of virucidal activity of chemical 
disinfectants used in the medical 
area - Test method and 
requirements (phase 2, step 2) 

This European Standard specifies a method 
for testing bactericidal activity by assessing 
reduction in the number of viable bacterial 
cells dried on a wood carrier under defined 
conditions. The approach can be applied to 
formulated products or to biocidal active 
substances. 

This European Standard specifies a method 
for testing fungicidal or yeasticidal activity 
by assessing reduction in the number of 
viable mould spores and/or yeast cells dried 
on a steel carrier under defined conditions. 
The approach can be applied to formulated 
products or to biocidal active substances. 

This European Standard specifies a method 
for testing bactericidal and/or yeasticidal 
activity by assessing reduction in the 
number of viable bacterial and/or yeast cells 
dried on a PVC carrier under defined 
conditions. The test applies to products that 
are used for disinfecting non-porous 
surfaces by wiping and includes ‘ready-to-
use wipes‘ which are impregnated with a 
microbicidal solution. 

This European Standard specifies a method 
for testing microbicidal activity of a 
disinfection process for the treatment of 
contaminated textile. The procedure is 
carried out by using a washing machine and 
microbicidal activity is assessed as the 
reduction in the number of viable test 
organisms, such as bacterial, mycobacterial 
or yeast cells and mould spores, dried on a 
cotton carrier under defined conditions. 

This European Standard specifies a method 
for testing virucidal activity by assessing 
reduction in the number of infectious virus 
particles dried on a steel carrier under 
defined conditions. The approach can be 
applied to formulated products or to biocidal 
active substances. 

Table 32: Other test methods and guidance documents 

Reference 

Title 

PT  Remarks 

ASTM 
E2196 

Standard Test Method for 
Quantification of Pseudomonas 
aeruginosa Biofilm Grown with 
Medium Shear and Continuous Flow 
Using Rotating Disk Reactor 

2,3, 
4 

This test method is used for growing a 
reproducible Pseudomonas aeruginosa biofilm 
in a continuously stirred tank reactor (CSTR) 
under medium shear conditions. In addition, 
the test method describes how to sample and 
analyse biofilm for viable cells. Available via: 
http://www.astm.org/Standard/ or the 
national standardisation bodies 

 
 
 
 
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Reference 

Title 

PT  Remarks 

ASTM 
E2274 

Standard Test Method for Evaluation 
of Laundry Sanitizers and 
Disinfectants 

2,3 

ASTM 
E2406 

Standard Test Method for Evaluation 
of Laundry Sanitizers and 
Disinfectants for Use in High 
Efficiency Washing Operations 

ASTM 
E2562 

Standard Test Method for 
Quantification of Pseudomonas 
aeruginosa Biofilm Grown with High 
Shear and Continuous Flow using 
CDC Biofilm Reactor 

2,3,
4 

DIN SPEC 
10534 

Food hygiene - Commercial 
dishwashing - Hygiene 
requirements, testing 

4 

This test method is designed to evaluate 
sanitizing/disinfectant laundry 
detergents/additives for use in top-loading 
automatic clothes washing operations. This 
test method is designed predominantly to 
provide testing with representative 
vegetative bacteria but can also be designed 
to accommodate the testing of fungi and 
viruses. 

This test method is designed to evaluate 
sanitizing/disinfectant laundry 
detergents/additives for use in high efficiency 
(HE) automatic clothes washing operations 
that typically utilize very low wash water 
volumes. This test method is designed to 
provide testing with representative 
vegetative bacteria but can also be designed 
to accommodate the testing of fungi and 
viruses. 

This test method specifies the operational 
parameters required to grow a reproducible 
Pseudomonas aeruginosa biofilm under high 
shear. The resulting biofilm is representative 
of generalized situations where biofilm exists 
under high shear rather than being 
representative of one particular environment. 
Available via: 
http://www.astm.org/Standard/ or the 
national standardisation bodies 

This document is a summary of the standards 
DIN 10510, DIN 10511, DIN 10512 and DIN 
10522. It specifies hygiene requirements 
relating to the design, construction and 
operation of commercial warewashers and in 
particular provides information on their 
hygienic and proper operation, on cleaning 
and disinfection of wash ware and on care 
and maintenance of the machinery. It 
describes the methods for testing hygienic 
operation. Available via: 
http://www.beuth.de/en/ or the national 
standardisation bodies 

3,4  DVG Guidelines specify methods for testing 

DVG 
Guidelines 

Guidelines for the testing of 
disinfection procedures and 
chemical disinfectants; 

Original title: Richtlinien für die 
Pruefung von 
Desinfektionsverfahren und 
chemischen Desinfektionsmitteln 

ISO/TS 
15883-5 

Washer-disinfectors - Part 5: Test 
soils and methods for demonstrating 
cleaning efficacy 

2,3,
4 

activty of chemical disinfectants against 
bacteria, yeasts and fungal spores, viruses, 
and parasites. They apply to the veterinary 
and the food sector, such as animal 
husbandry, veterinary practices, meat 
production/food of animal origin, and large-
scale/canteen kitchens (except ward kitchens 
catering patients). DVG Guidelines are 
published by the German Veterinary Medical 
Society (DVG). Available in German via:  
http://www.desinfektion-dvg.de 

ISO 15883 relates to a series of standards 
that specify the required performance levels 
of Washer-Disinfectors. Part 5, the Technical 
Specification (TS), describes a method to 
generate biofilm formed by Pseudomonas 
aeruginosa. Available via: 
http://www.iso.org/iso/home.htm or the 
national standardisation bodies 

 
 
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356 

Reference 

Title 

PT  Remarks 

NF T72-
281 

Methods of airborne disinfection of 
surfaces - Determination of 
bactericidal, fungicidal, yeasticidal, 
mycobactericidal, tuberculocidal, 
sporicidal and virucidal activity, 
including bacteriophages; 

Original title: Procédés de 
désinfection des surfaces par voie 
aérienne - Détermination de 
l'activité bactéricide, fongicide, 
levuricide, mycobactéricide, 
tuberculocide sporicide et virucide 
incluant les bactériophages 

2,3,
4 

This French standard specifies a method for 
testing microbicidal activity of airborne 
disinfection processes. The tested product is 
diffused, e.g. in gaseous form or as an 
aerosol, to reduce the number of relevant 
test organisms, such as bacteria, bacterial 
spores, yeasts, and fungal spores. Available 
in French via: http://www.afnor.org/en or the 
national standardisation bodies 

Nordic 
Working 
Paper 

Efficacy Assessment of Treated 
Articles: A guidance 

1,2,
3,4 

OECD 
Series on 
Biocides 
No. 1 

Guidance Document on the 
Evaluation of the Efficacy of 
Antimicrobial Treated Articles with 
Claims for External Effects 

OECD 
Series on 
Biocides 
No. 4  

Guidance Document for 
Demonstrating Efficacy of Pool and 
Spa Disinfectants and Field Testing 
(Series on Testing and Assessment 
No. 170 and Series on Biocides No. 
4) 

2 

OECD 
Series on 
Biocides 
No. 6 

Guidance Document on Quantitative 
Methods for Evaluating the Activity 
of Microbiocides used on Hard Non-
Porous Surfaces (Series on Testing 
and Assessment No. 187 and Series 
on Biocides No. 6). 

2 
(4) 

OECD 
Series on 
Biocides 
No. 8  

Guidance Document for Quantitative 
Method for Evaluating Antibacterial 
Activity of Porous and Non-Porous 
Antibacterial Treated Materials 
(Series on Testing and Assessment 
No. 202 and Series on Biocides No. 
8) 

1,2,
3,4 

The document provides guidance on efficacy 
testing of biocides used in treated articles. 
The presence and relevance of existing 
standard test methods is described and, 
where they do not exist or where they do not 
provide sufficient support, the nature of the 
data required will be described. The 
document was published by the Nordic 
Council of Ministers. Open access via: 
http://www.norden.org/en/publications/publi
kationer/2014-904/ 

The document guidance on efficacy testing of 
articles treated with antimicrobials and 
articles modified to exert an antimicrobial 
effect.  
http://www.oecd.org/env/ehs/pesticides-
biocides/41692131.pdf 

The document provides guidance on setting 
up a strategy for efficacy testing of pool and 
spa disinfectants in a laboratory scale testing 
phase and a field testing phase in a full-size 
swimming or spa pool. Open access via: 
http://www.oecd.org/env/ehs/pesticides-
biocides/biocidestestguidelinesandguidencedo
cuments.htm 

This document describes four quantitative 
methods for testing bactericidal, 
mycobactericidal, fungicidal and virucidal 
activity on steel carriers with high application 
volumes of liquid products. Open access via: 
http://www.oecd.org/env/ehs/pesticides-
biocides/biocidestestguidelinesandguidencedo
cuments.htm 

The document provides guidance for testing 
the basic antibacterial performance of porous 
(textile) and non-porous (plastic) materials 
that have been treated with a biocide with 
the intention of introducing 
antibacterial/hygienic properties into that 
material. Open access via: 
http://www.oecd.org/env/ehs/pesticides-
biocides/biocidestestguidelinesandguidencedo
cuments.htm  

 
 
 
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   357 

Reference 

Title 

PT  Remarks 

VAH 
Standard 
methods 

VAH certification of chemical 
disinfection procedures; 

1,2 

Original title: VAH-Zertifizierung 
chemischer Desinfektionsverfahren 

VAH Standard methods specify methods for 
testing activty of chemical disinfectants 
against bacteria (incl. mycobacteria), yeasts, 
and fungal spores. They apply to testing 
products used for disinfection in public 
facilities (medical and other) and, in the 
event of substantiated medical indications, 
also in the private home. VAH Standard 
methods are published by the Association for 
Applied Hygiene (VAH). Available in German 
via: http://www.mhp-verlag.de/en/home/  

 
 
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Appendix 3. Table of Reference Test Organisms (PT 1-5) 

This  table  (Table  33)  is  given  as  a  general  overview  of  relevant  test  organisms  for  testing 
disinfectants in accordance with the BPR. 

This table comprises mainly those reference test organisms that are included in the EN norms 
covered  by  EN14885.  Furthermore,  strains  are  listed  that  are  recommended  for  some  uses 
(e.g. endoparasites from DVG standard). 

The  reader  can  check  the  website  of  the  CEN  (European  Standardization  Organizations): 
www.cen.eu for new and updated standards. 

Since  the  EN  systematics  of  WG’s  1  to  3  does  not  fit  exactly  to  the  BPR  PT  scheme,  in 
borderline cases an indicated  reference test organism might be used for  other PTs as well. In 
cases  where  there  are  discrepancies  between  this  ECHA  guidance  and  the  guidance  in  EN 
14885,  the  ECHA  Guidance  should  be  followed  as  the  leading  guidance.  However,  EN  14885 
can be followed with satisfactory justification to meet the requirements of the BPR. 

Tests  with  test  organisms  in  addition  to  those  mentioned  below  are  acceptable,  if  adequate 
scientific evidence is submitted on which the relevance of the test organism to the field of use 
can be judged. 

Key for Table 33:  

* X = basic requirement to claim activity against micro-organism; 

(X) = basic requirement for specific use as described in the table below in brackets; 

O = optional; 

**  the  strain  ATTC  16404  was  previously  classified  as  Aspergillus  niger  but  after 
reclassification in 2008 it is now classified as Aspergillus brasiliensis; 

*** for a limited spectrum virus claim in PT1 Poliovirus does not have to be tested; 

**** in EN suspension tests efficacy against enteroviruses and norovirus should be tested. 

PT1*  PT2*  PT3*  PT4*  PT5* 

Table 33: Reference Test Organisms 

Micro-organisms 

Bacteria  

Staphylococcus aureus ATCC 6538 

Pseudomonas aeruginosa ATCC 15442 (not for teat disinfection) 

Enterococcus hirae ATCC 10541 (not for teat disinfection) 

Escherichia  coli  ATCC  10536  (PT2:  domestic  area  and  industry;  PT3 
teat disinfection) 

X 

X 

X 

X 

X 

X 

X 

X 

X 

(X) 

(X) 

Escherichia coli K12 NCTC 10538 (PT2 medical area) 

X 

(X) 

Escherichia coli A3 (simulated use test) 

Salmonella Typhimurium ATCC 13311 

Lactobacillus brevis DSM6235 

Enterobacter cloacae DSM 6234 

Enterococcus faecium ATCC 6057 (for T ≥40°C) 

Proteus vulgaris ATCC 13315 (not for teat disinfection) 

Enterococcus faecium Teltow 11 (simulated use test) 

Proteus vulgaris ATCC 13315 (not for teat disinfection) 

O 

O 

O 

(X) 

X 

X 

X 

X 

X 

X 

O 

O 

O 

(X) 

X 

X 

X 

X 

(X) 

(X) 

Streptococcus uberis ATCC 19436 (teat disinfection) 

(X) 

O 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Micro-organisms 

PT1*  PT2*  PT3*  PT4*  PT5* 

Legionella  pneumophila  ATCC  33152  (PT2:  pools,  hot  tubs;  PT4: 
drinking water systems, PT5: in collective drinking water systems) 

Legionella pneumophila ATCC 43108 

Yeasts 

(X) 

O 

Candida albicans ATCC 10231 

X 

X 

X 

Saccharomyces cerevisiae ATCC 9763 (breweries) 

Saccharomyces cerevisiae DSM 70487 (breweries) 

Fungal spores 

X 

O 

O 

X 

(X) 

(X) 

Aspergillus brasiliensis** ATCC 16404  

X 

X 

X 

X 

O 

Viruses 

Polio virus type 1, LSc-2ab (Picornavirus) 

X***  X 

Adenovirus, type 5, strain Adenoid 75, ATCC VR-5. 

Murine norovirus, strain S99 Berlin 

Murine Parvovirus, strain Crawford, ATCC VR-1346 (for T ≥40°C)  

Bovine Enterovirus Type 1, ECBO - Virus ATCC VR-248 

Rotavirus (pools, hot tubs) 

Enterovirus, e.g. Coxsackievirus B4 or B5 

Enveloped Viruses 

X 

X 

X 

X 

(X) 

(X) 

X 

X**** 

X**** 

X 

X 

(X) 

MVA = Modified Vacciniavirus Ankara (teat disinfection) 

X 

(X) 

Bacteriophages 

Bacteriophage P001 DMS 4262 (milk industry) 

Bacteriophage P008 DMS 10567 (milk industry) 

Bacteriophage  MS2  DSM  13767  or  ATCC  15597-B1™  (simulated 
use test) 

Bacteriophage PRD1 DSM 19107 (simulated use test) 

Mycobacteria  

Mycobacterium terrae ATCC 15755 

Mycobacterium avium ATCC 15769 

(PT1  and PT2 claim  for mycobactericidal: both, tuberculocidal:  M. 
terrae only) 

Bacterial spores 

Spores of Bacillus cereus ATCC 12826 (bee hives) 

Spores of Bacillus subtilis ATCC 6633 (bee hives) 

Spores of Clostridium sporogenes ATCC 7955  

Spores of Geobacillus stearothermophilus (for T ≥60°C) 

Endoparasites 

X 

X 

X 

X 

O 

X 

O 

O 

X 

(X) 

O 

O(X)  X 

O 

O 

Oocysts of Eimeria tenella strain Houghton (chicken farms) 

(X) 

X 

(X) 

(X) 

X 

X 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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360 

Appendix 4. Overview  of  standards,  test  conditions  and pass 
criteria (PT 1-5) 

The overview is presented in a number of tables.  

These  tables  provide  an  overview  of  available  phase  2,1  and  2,2  EN  standards  which  are 
applicable for testing the efficacy of disinfectant biocides. This overview is not exhaustive. For 
other  or  more  specific  uses  and  tests  other  than  EN  standards,  reference  should  be  made  to 
the relevant sections of this guidance. 

It should be noted that although this Guidance is mainly based on EN standards, there are 
some cases where there are discrepancies amongst the EN tests and in such cases the ECHA 
Guidance should be followed as the leading guidance. Where noted these are identified in the 
table. 

The reader is strongly advised to check whether there are new versions of the standards on the 
website of the CEN: www.cen.eu. 

It should be noted that if tests other than CEN standards (notably when no CEN tests are 
available) are used, and pass criteria are available, these should be met (unless stated 
differently in this guidance). When the test does not provide pass criteria, the criteria in this 
table can be taken into account as guidance for what level of reduction is normally required.  

In all cases, deviations from these standards are possible but should be justified in the 
application. 

 
 
 
 
 
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Required lg 
reduction 

Requirements1 

Test required2 

Contact time3 

Temp (°C) Soiling conditions4 

PT 1 
Product type / micro-
organism 

PT 1 hygienic handrub 

bacteria 

bacteria 

yeast 

PT 1 hygienic handwash 

bacteria 

bacteria 

yeast 

Basic requirement - 2,1 test  EN 13727 / EN 12765 

30 - 60 sec6 

20 

clean / dirty 

5 

Basic requirement - 2,2 test  EN 1500 

30 - 60 sec6 

skin T 

none 

≥ propan-2-ol7 

Basic requirement - 2,1 test  EN 13624 / EN 16505 

30 - 60 sec6 

mycobacteria / tuberculosis 

Optional - 2,1 test 

EN 14348 

viruses 

Optional - 2,1 test 

EN 14476 

30 - 60 sec6 

30 - 120 sec6 

fungal spores 

Optional - 2,1 test 

EN 13624 / EN 16505 

30 - 60 sec6 

20 

20 

20 

20 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

4 

4 

4 

4 

Basic requirement - 2,1 test  EN 13727 / EN 12765 

30 - 60 sec6 

20 

dirty8 

3 / 59 

Basic requirement - 2,2 test  EN 1499 

30 - 60 sec6 

skin T 

none 

> control10 

Basic requirement - 2,1 test  EN 13624 / EN 16505 

30 - 60 sec6 

mycobacteria / tuberculosis 

Optional - 2,1 test 

EN 14348 

30 - 60 sec6 

viruses 

Optional - 2,1 test 

EN 14476 

30 - 120 sec6 

fungal spores 

Optional - 2,1 test 

EN 13624 / EN 16505 

30 - 60 sec6 

PT 1 surgical hand disinfection 

20 

20 

20 

20 

dirty8 

dirty8 

dirty8 

dirty8 

2 / 49 

4 

4 

2 / 49 

Basic requirement - 2,1 test  EN 13727 

2-3 min11 

20 

clean / dirty 

5 

bacteria 

bacteria 

yeast 

Basic requirement - 2,2 test  EN 12791 

Basic requirement - 2,1 test  EN 13624 

mycobacteria / tuberculosis 

Optional - 2,1 test 

viruses 

fungal spores 

Optional - 2,1 test 

Optional - 2,1 test 

EN 14348 

EN 14476 

EN 13624 

2-3 min11 

2-3 min11 

2-3 min11 

2-3 min11 

2-3 min11 

skin T 

none 

≥ propan-1-ol12 

20 

20 

20 

20 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

4 

4 

4 

4 

 
 
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PT 2 
Product type / micro-
organism 

Requirements1 

Test required2 

Contact time3 

Temp 
(°C) 

Soiling 
conditions4 

Required lg 
reduction 

PT 2 hard surfaces and other uses where EN tests are applicable, use in healthcare 

bacteria 

bacteria 

yeast 

yeast 

Basic requirement - 2,1 test  EN 13727 / EN 12765 

5 min13 / 60 min  20 

clean / dirty 

5 

Basic requirement - 2,2 test  EN 13697 / EN 1661514  5 min13 / 60 min  20 

clean / dirty 

4 / 5 

Basic requirement - 2,1 test  EN 13624 / EN 16505 

5 min13 / 60 min  20 

clean / dirty 

4 

Basic requirement - 2,2 test  EN 13697 / EN 1661514  5 min13 / 60 min  20 

clean / dirty 

3 / 4 

mycobacteria / tuberculosis 

Optional - 2,1 test 

EN 14348 

5 min13 / 60 min  20 

clean / dirty 

viruses 

viruses 

fungal spores 

fungal spores 

Optional - 2,1 test 

EN 14476 

5 min13 / 60 min  20 

clean / dirty 

Optional – 2,2 test 

See15 

5 min13 / 60 min  20 

clean / dirty 

Optional - 2,1 test 

EN 13624 / EN 16505 

5 min13 / 60 min  20 

clean / dirty 

Optional - 2,2 test 

EN 13697 

5 min13 / 60 min  20 

clean / dirty 

PT 2 hard surfaces and other uses where EN tests are applicable, use other than in healthcare 

bacteria 

bacteria 

yeast 

yeast 

Basic requirement - 2,1 test  EN 13727 / EN 12765 

5 min13 / 60 min  20 

clean / dirty 

Basic requirement - 2,2 test  EN 13697 / EN 1661514  5 min13 / 60 min  20 

clean / dirty 

4 / 5 

Optional - 2,1 test 

EN 13624 / EN 16505 

5 min13 / 60 min  20 

clean / dirty 

4 

Optional - 2,2 test 

EN 13697 / EN 1661514  5 min13 / 60 min  20 

clean / dirty 

3 / 4 

mycobacteria / tuberculosis 

Optional - 2,1 test 

EN 14348 

5 min13 / 60 min  20 

clean / dirty 

viruses 

viruses 

fungal spores 

fungal spores 

Optional - 2,1 test 

EN 14476 

5 min13 / 60 min  20 

clean / dirty 

Optional – 2,2 test 

See15 

5 min13 / 60 min  20 

clean / dirty 

Optional - 2,1 test 

EN 13624 / EN 16505 

5 min13 / 60 min  20 

clean / dirty 

Optional - 2,2 test 

EN 13697 

5 min13 / 60 min  20 

clean / dirty 

4 

4 

4 

4 

3 

4 

4 

4 

4 

3 

5 

   
 
 
 
 
 
 
 
Basic requirement - 2,1 test  EN 13727 / EN 12765 

as claimed 

20 

clean / dirty16 

Guidance on the BPR: Volume II Parts B+C  
Version 4.0 December 2021 

PT 2 room disinfection (including use in healthcare) 

bacteria 

bacteria 

yeast 

yeast 

Basic requirement - semi- field 
trial 
Basic requirement - 2,1 test  EN 13624 / EN 16505 

NF T 72-28117 

Basic requirement - semi- field 
trial 

NF T 72-28117 

as claimed 

as claimed 

as claimed 

as claimed 

mycobacteria / tuberculosis 

Optional - 2,1 test 

EN 14348 

mycobacteria / tuberculosis 

Optional - semi-field trial 

NF T 72-28117 

as claimed 

viruses 

viruses 

Optional - 2,1 test 

EN 14476 

as claimed 

Optional - semi-field trial 

NF T 72-28117 

as claimed 

fungi, fungal spores 

Optional - 2,1 test 

EN 13624 / EN 16505 

as claimed 

fungi, fungal spores 

Optional - semi-field trial 

NF T 72-28117 

as claimed 

PT 2 (instrument) disinfection by immersion or filling 

bacteria 

bacteria 

yeast 

yeast 

viruses 

viruses 

fungal spores 

fungal spores 

Basic requirement - 2,1 test  EN 13727 

Basic requirement - 2,2 test  EN 14561 

Basic requirement - 2,1 test  EN 13624 

Basic requirement - 2,2 test  EN 14562 

Basic requirement - 2,1 test18  EN 14476 

Optional – 2,2 test 

See15 

Basic requirement - 2,1 test18  EN 13624 

Basic requirement - 2,2 test18  EN 14562 

mycobacteria / tuberculosis 

Optional - 2,1 test 

mycobacteria / tuberculosis 

Optional - 2,2 test 

EN 14348 

EN 14563 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

clean / dirty16 

clean / dirty16 

clean / dirty16 

clean / dirty16 

clean / dirty16 

clean / dirty16 

clean / dirty16 

clean / dirty16 

clean / dirty16 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

363 

5 

5 

4 

4 

4 

4 

4 

4 

4 

4 

5 

5 

4 

4 

4 

4 

4 

4 

4 

4 

 
 
364 

PT 2 textiles 

bacteria 

bacteria 

yeast 

yeast 

Basic requirement - 2,1 test  EN 13727 / EN 12765 

as claimed 

Basic requirement - 2,2 test 

EN 16616 / ASTM E2406 / 
ASTM E227421 

as claimed 

Basic requirement - 2,1 test  EN 13624 / EN 16505 

as claimed 

Basic requirement - 2,2 test 

EN 16616 / ASTM E2406 / 
ASTM E227421 

as claimed 

mycobacteria / tuberculosis 

Optional - 2,1 test 

EN 14348 

as claimed 

mycobacteria / tuberculosis 

Optional - 2,2 test 

EN 16616 / ASTM E2406 / 
ASTM E227421 

as claimed 

viruses 

viruses 

Optional - 2,1 test 

EN 14476 

as claimed 

Optional - 2,2 test 

EN 16616 / ASTM E2406 / 
ASTM E227421 

as claimed 

fungal spores 

Optional - 2,1 test 

EN 13624 / EN 16505 

as claimed 

fungal spores 

Optional - 2,2 test 

EN 16616 / ASTM E2406 / 
ASTM E227421 

as claimed 

Guidance on the BPR: Volume II Parts B+C  
Version 4.0 December 2021 

as 
claimed19 
as 
claimed19 
as 
claimed19 
as 
claimed19 

as 
claimed19 

as 
claimed19 

clean / dirty20 

5 

clean / dirty20 

7 / 4 / 4 

clean / dirty20 

4 

clean / dirty20 

6 / 3 / 3 

clean / dirty20 

4 

clean / dirty20 

7 / 4 / 4 

clean / dirty20 

as 
claimed19 
as 
claimed19  clean / dirty20 
as 
clean / dirty20 
claimed19 
as 
claimed19 

clean / dirty20 

4 

4 

6 / 3 / 3 

   
 
 
 
 
 
 
 
 
 
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Requirements1 

Test required2 

Contact time3 

Temp 
(°C) 

Soiling conditions4 

Required lg 
reduction 

PT 3 

Product type / micro-
organism 

PT 3 hard surfaces 

bacteria 

bacteria 

yeast 

yeast 

Basic requirement - 2,1 test 

EN 1656 

30 min22 

Basic requirement - 2,2 test 

EN 14349 / EN 16437 

30 min22 

Basic requirement - 2,1 test 

EN 1657 

Basic requirement - 2,2 test 

EN 1643823 

10 

10 

10 

10 

10 

10 

10 

10 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

5 

4 

4 

3 

4 

3 

4 

4 

30 min22 

30 min22 

30 min22 

30 min22 

30 min22 

30 min22 

fungal spores 

Optional - 2,1 test 

EN 1657 

fungal spores 

Optional - 2,2 test 

EN 1643823 

viruses 

Optional - 2,1 test 

mycobacteria / tuberculosis  Optional - 2,1 test 

endoparasites 

Optional 

PT 3 hard surfaces in transport vehicles 

EN 14675 

EN 14204 

DVG 

Bacteria, yeasts, fungal 
spores, mycobacteria / 
tuberculosis, endoparasites 

As PT 3 hard surfaces 

5 min22 

As PT 3 hard surfaces 

viruses 

Basic requirement - 2,1 test 

EN 14675 

5 min22 

10 

clean / dirty 

4 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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PT 3 teat disinfection 

bacteria pre-milking 

Basic requirement - 2,1 test 

EN 1656 

bacteria post-milking 

Basic requirement - 2,1 test 

EN 1656 

30-60 sec24 

5 min24 

bacteria pre-milking 

Basic requirement - 2,2 test 

to be developed 

30-60 sec24 

bacteria post-milking 

Basic requirement - 2,2 test 

to be developed 

5 min24 

yeast pre-milking 

Basic requirement - 2,1 test 

EN 1657 

yeast post-milking 

Basic requirement - 2,1 test 

EN 1657 

yeast pre/post-milking 

Optional – 2,2 test 

See25 

mycobacteria / tuberculosis 
pre/post milking 
fungal spores pre/post 
milking 
fungal spores pre/post 
milking 

Optional - 2,1 test 

EN 14204 

Optional - 2,1 test 

EN 1657 

Optional - 2,2 test 

EN 16438 

viruses pre/post milking 

Optional - 2,1 test 

EN 14675 

algae 

Optional 

no test available 

PT 3 hoof disinfection 

bacteria 

bacteria 

Basic requirement - 2,1 test26  EN 1656 

Basic requirement - 2,2 test26  EN 16437 

yeast / fungal spores 

Optional - 2,1 test26 

EN 1657 

yeast / fungal spores 

Optional - 2,2 test26 

EN 1643823 

viruses 

Optional - 2,1 test26 

EN 14675 

mycobacteria / tuberculosis  Optional - 2,1 test26 

EN 14204 

30-60 sec24 

5 min24 

30-60 sec / 5 
min24 
30-60 sec / 5 
min24 
30-60 sec / 5 
min24 
30-60 sec / 5 
min24 

5 min 

5 min 

5 min 

5 min 

5 min 

5 min 

30 

30 

30 

30 

30 

30 

30 

30 

30 

30 

10 

10 

10 

10 

10 

10 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

dirty27 

dirty27 

dirty27 

dirty27 

dirty27 

dirty27 

5 

4 

4 

3 

4 

3 

4 

4 

5 

4 

4 

3 

4 / 5 

4 

   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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PT 3 disinfection of hatching-eggs 

bacteria 

bacteria 

Basic requirement - 2,1 test 

EN 1656 

Basic requirement - 2,2 test 

EN 16437 

fungal spores 

Basic requirement - 2,1 test 

EN 1657 

fungal spores 

Basic requirement - 2,2 test 

No standard guideline 
available28 

other target organisms 

Optional - 2,1 test 

other target organisms 

Optional - 2,2 test 

PT 3 textile disinfection 

as claimed 

as claimed 

as claimed 

as claimed 

30 

30 

30 

30 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

As PT 3 porous surfaces 

As PT 3 porous surfaces 

bacteria 

bacteria 

yeasts 

yeasts 

Basic requirement - 2,1 test 

EN 1656 

Basic requirement - 2,2 test 

Basic requirement - 2,1 test 

Basic requirement - 2,2 test 

EN 16616 / ASTM 
E240621 
EN 1657 

EN 16616 / ASTM 
E240621 

as claimed 

as claimed 

as claimed 

as claimed 

as 
claimed19 
as 
claimed19 
as 
claimed19 
as 
claimed19 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

other target organisms 

Optional - 2,1 test 

other target organisms 

Optional - 2,2 test 

PT 3 disinfection of beehives and beekeeping equipment 

As PT 2 textile, with PT 3 soiling 

As PT 2 textile, with PT 3 soiling 

bacteria 

bacteria 

Basic requirement - 2,1 test 

EN 1656 

Basic requirement - 2,2 test 

EN 16437 

bacterial spores 

Basic requirement - 2,1 test 

EN 13704 

as claimed 

as claimed 

as claimed 

bacterial spores 

Basic requirement - 2,2 test 

EN 16437 adapted 

as claimed 

10 

10 

10 

10 

clean/dirty 

clean/dirty 

clean/dirty 

clean/dirty 

other target organisms 

Optional - 2,1 test 

other target organisms 

Optional - 2,2 test 

As PT 3 porous surfaces 

As PT 3 porous surfaces 

5 

4 

4 

3 

5 

7 / 4 

4 

6 / 3 

5 

4 

4 

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Requirements1 

Test required2 

Contact time3 

Temp  

Soiling conditions4 

Required lg 
reduction 

368 

PT 4 

Product type / micro-
organism 

PT 4 hard surfaces 

bacteria 

bacteria 

yeast 

yeast 

Basic requirement - 2,1 test 

EN 1276 

Basic requirement - 2,2 test 

EN 13697 

Basic requirement - 2,1 test 

EN 1650 

Basic requirement - 2,2 test 

EN 13697 

mycobacteria 

Optional - 2,1 test 

EN 14348 

viruses 

viruses 

Optional - 2,1 test 

EN 1447629 

Optional – 2,2 test 

See15 

bacteriophages 

Optional - 2,1 test 

EN 1361029 

fungal spores 

Optional - 2,1 test 

fungal spores 

Optional - 2,2 test 

bacterial spores 

Optional - 2,1 test 

PT 4 inner surfaces without circulation 

EN 1650 

EN 13697 

EN 13704 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

as claimed 

20 

20 

20 

20 

20 

20 

20 

20 

20 

20 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

clean / dirty 

as claimed 

as claimed30  

5 

4 

4 

3 

4 

4 

4 

4 

3 

3 

5 

4 

see PT04 hard surfaces 

PT 4 inner surfaces by CIP 

bacteria 

yeast 

Basic requirement - 2,1 test 

EN 1276 

as claimed 

as claimed30 clean / dirty 

Basic requirement - 2,1 test 

EN 1650 

as claimed 

as claimed30 clean / dirty 

other target organisms 

see PT 4 hard surfaces 

   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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PT 4 surfaces in drinking water systems 

bacteria 

Legionella 

Basic requirement - 2,1 test 

EN 1276 

Optional - 2,1 test 

EN 13623 

as claimed 

as claimed 

20 

20 

clean / dirty 

clean / dirty 

other organisms when 
claimed 

Optional - 2,1 test 

as PT 4 hard surfaces 

Legionella 

bacteria 

Basic requirement field trial 

See 5.4.4.6.2 section Field trials 

Optional - field trial 

See 5.4.4.6.2 section Field trials 

PT 4 equipment disinfection by soaking 

bacteria 

bacteria 

yeast 

yeast 

Basic requirement - 2,1 test 

EN 1276 

as claimed 

as claimed  clean / dirty 

Basic requirement - 2,2 test 

EN 13697 

as claimed 

as claimed  clean / dirty 

Basic requirement - 2,1 test 

EN 1650 

as claimed 

as claimed  clean / dirty 

Basic requirement - 2,2 test 

EN 13697 

as claimed 

as claimed  clean / dirty 

other organisms when 
claimed 
other organisms when 
claimed 
PT 4 surfaces in veterinary water systems 

Optional - 2,1 test 

Optional - 2,1 test 

as PT 4 hard surfaces 

as PT 4 hard surfaces 

bacteria 

bacteria 

Basic requirement - 2,1 test 

EN 1276 

as claimed 

Basic requirement - 2,2 test 

EN 13697 

as claimed 

20 

20 

clean / dirty 

clean / dirty 

other organisms when 
claimed 

Optional - 2,1 test 

as PT 4 hard surfaces 

5 

4 

5 

4 

4 

3 

5 

4 

 
 
 
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PT 4 disinfection in dishwashing machines and crate washers 

bacteria 

bacteria 

bacteria 

yeast 

yeast 

yeast 

other 
claimed 
other 
claimed 
other 
claimed 

Basic requirement - 2,1 test 

EN 1276 

as claimed 

as claimed19 clean / dirty 

Basic requirement - 2,2 test 

EN 13697 

as claimed 

as claimed19 clean / dirty 

Basic requirement - 3 test 

DIN SPEC 10534 

as claimed 

as claimed19 clean /dirty 

Basic requirement - 2,1 test 

EN 1650 

as claimed 

as claimed19 clean / dirty 

Basic requirement - 2,2 test 

EN 13697 

as claimed 

as claimed19 clean / dirty 

5 

4 

4 

3 

Basic requirement - 3 test 

DIN SPEC 10534 

as claimed 

as claimed19 clean / dirty 

organisms 

organisms 

organisms 

when 

Basic requirement - 2,1 test 

when 

Basic requirement - 2,2 test 

when 

Basic requirement - 3 test 

as PT 4 hard surfaces 

as PT 4 hard surfaces 

According to DIN SPEC 10534 

   
 
 
 
 
 
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PT 5 

Product type / micro-
organism 

Requirements1 

Test required2 

Contact time3  Temp  

PT 5 Drinking water suppliers and their water distribution systems 

bacteria 

bacteria 

viruses 

viruses 

Basic requirement – 2,1 test 

EN 1276 adapted 

max 30 min 

Basic requirement – 
simulated use test 

Test protocol31 

10 min / 25 
min 

Basic requirement – 2,1 test 

EN 14476 adapted 

max 30 min 

Basic requirement – 
simulated use test 

Test protocol31 

10 min / 25 
min 

other organisms 

Optional – 2,1 test 

PT 5 Raw water for individual supply (1-2 premises) 

bacteria 

bacteria 

viruses 

viruses 

Basic requirement – 2,1 test 

EN 1276 adapted 

max 30 min 

Basic requirement – 
simulated use test 

Test protocol31 

10 min / 25 
min 

Basic requirement – 2,1 test 

EN 14476 adapted 

max 30 min 

Basic requirement – 
simulated use test 

Test protocol31 

10 min / 25 
min 

other organisms 

Optional – 2,1 test 

PT 5 Collective drinking water systems 

bacteria 

Legionella 

Legionella 

Legionella 

Basic requirement –2,1 test 

EN 1276 adapted 

25 min 

Basic requirement – 2,1 test 

EN 13623 

25 min 

Optional – FR Method 
(simulated use) 

as claimed 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

Soiling 
conditions4 

Required 
lg 
reduction 

clean/dirty 

5 

2 mg DOC / L 

clean/dirty 

2 mg DOC / L 

dirty 

2 mg DOC / L 

dirty 

2 mg DOC / L 

2 / 4 

4 

2 / 4 

5 

2 / 4 

4 

2 / 4 

clean 

clean 

2 mg DOC / L 

5 

4 

4 

Basic requirement – Field test 

See Guidance: Vol II B+C, section 5.4.5.4.2 (Test conditions/Field Trials) 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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PT 5 Water in reservoirs 

bacteria 

Basic requirement – 2,1 test 

EN 1276 adapted 

as claimed 

15 

clean/dirty 

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bacteria 

Basic requirement – 
Simulated use test 

Test to be developed 

as claimed 

15 

clean/dirty 

criteria for 
drinking 
water 
according to 
the Drinking 
water 
directive 
should be 
met 

viruses 

Basic requirement – 2,1 test 

EN 14476 adapted 

as claimed 

15 

clean/dirty 

4 

viruses 

Basic requirement – 
Simulated use test 

Test to be developed 

as claimed 

15 

clean/dirty 

criteria for 
drinking 
water 
according to 
the Drinking 
water 
directive 
should be 
met 

other organisms (e.g 
Legionella) 
PT 5 Water of undefined quality for small scale use (up to 5 L/person/day) 

Optional – Simulated use test 

bacteria 

viruses 

Basic requirement – 2,1 test 

EN 1276 adapted 

max 30 min 

Basic requirement – 2,1 test 

EN 14476 adapted 

max 30 min 

15 

15 

dirty  

dirty 

5 

4 

other organisms 

Optional – 2,1 test 

all organisms 

Optional field trial including 
turbidity 

max 30 min, 
unless a longer 
CT is justified 
max 30 min, 
unless a longer 
CT is justified 

   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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PT 5 Water for animals 

bacteria 

bacteria 

other organisms 

Optional – 2,1 test modified 

other organisms 

Optional – simulated use test 
or Field test 

NOTES on TABLES 

Basic requirement – 2,1 test 

EN 1276 adapted 

max 30 min32 

15 

clean/dirty 

5 

Basic requirement – 
simulated use test or Field 
test 

Test protocol31 or field 
trial see 5.4.5.7.2 

10 min / 25 
min or field trial 
as claimed 

clean/dirty 

15 

15 

2 / 4 or field 
trial see 
5.4.5.7.2 
According to 
test 

1  Requirements:  basic  requirements  are  mandatory  and  have  to  be  fulfilled  for  authorisation  of  a  product  with  this  intended  use.  In 
addition,  other  organisms  claimed  are  optional,  i.e.  if  the  requirements  for  these  organisms  are  not  fulfilled  these  organisms  will  be 
excluded from the claim. 

2  EN-tests  are  strongly  advised  but  not  mandatory.  Other  tests  carried  out  according  to  standard  guidelines  are  acceptable  if  a  clear 
description of the test procedure (including contact time, soiling, temperature, suitable controls, log10 reduction, etc.) and justification is 
provided. 

3  Contact  time:  maximum  acceptable  contact  times  are  stated,  at  which  efficacy  should  be  demonstrated.  If  a  shorter  contact  time  is 
stated on the label, efficacy has to be demonstrated at this shorter contact time. It is recommended to only use contact times mentioned 
in the EN standards as obligatory or additional contact time, to keep the robustness of the test as much as possible. 

4 Soiling conditions: low level soiling conditions are acceptable if it is stated on the label that cleaning prior to disinfection is necessary. 
Otherwise, and in case no prior cleaning is possible, dirty conditions have to be included in the tests. 

PT 1 and 2 For hospitals and health care: 

Dirty 3 g/L bovine albumin + 3 ml/L sheep erythrocytes // Clean 0.3 g/L bovine albumin 

PT 1 and 2 other uses: 

Dirty 3 g/L bovine albumin // Clean 0.3 g/L bovine albumin 

PT 2  

cosmetic industry: 
Dirty 3 g/L bovine albumin and 5 g/L sodium dodecyl sulphate in separate tests 

PT 2  

surfaces in agricultural area (no plant protection claim): 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Dirty 10 g/L bovine albumin + 10 g/L yeast extract // Clean 3 g/L bovine albumin 

PT 3   general hard surface disinfectants, hoof and animal skin disinfection, pre-milking teat disinfection, and eggs in hatcheries: 

Dirty 10 g/L bovine albumin + 10 g/L yeast extract // Clean 3 g/L bovine albumin 

PT 3 

PT 3  

PT3 

outer surfaces of milking equipment 
Clean/Dirty 10 g/L skimmed milk 

teat disinfection 
pre milking: Dirty 10 g/L bovine albumin + 10 g/L yeast extract // Clean 3 g/L bovine albumin (different from EN 14885) 
post milking: Clean/Dirty 10 g/L skimmed milk 

textiles: 
depending  on  the  use,  either  milk  soiling  (see  teat  disinfection)  or  veterinary  soiling  (see  PT  3  general)  would  be  the 
relevant  type  of  soiling.  However,  since  the  phase  2,  step  2  test  for  textile  are  not  validated  for  this  type  of  soiling, 
consultation with CEN is needed. For the time  being, it is recommended to use the  obligatory interfering substance  in EN 
16616: sterile defibrinated sheep blood (12.5 ml sheep blood per kg textile) 

PT 4   general disinfection in food industry and other areas with surfaces in contact with food: 

Dirty 3 g/L bovine albumin // Clean 0.3 g/L bovine albumin 

PT 4   milk industry and milking equipment on farms: 

10 g/L skimmed milk 

PT 4   meat industry: 

Dirty 3 g/L bovine albumin + 3 ml/L sheep erythrocytes (different from EN 14885) 

PT 5   general: 

Dirty ≥ 15 mg DOC/L // Clean  >2 mg DOC/L. To realize this DOC (Dissolved Organic Carbon) either yeast extract or BSA 
can be used: use of other interfering substances should be justified. The DOC should be measured before adding the test 
product, and adjusted with yeast extract or BSA to reach the required DOC/L 
For EN13623 the standard soiling of the test can be used or the test can be adapted with the soiling stated above. 
For simulated-use test according to test protocol31 only the standard soiling of the test is used (i.e. 2 mg DOC/L) 

PT 5   drinking water suppliers and water distribution system 

primary disinfection: Dirty 15 mg DOC/L 
secondary disinfection: Clean 2 mg DOC/L 
raw water for individual supply , water of undefined quality: Dirty 15 mg DOC/L 

PT 5  

PT 5   drinking water in collective drinking water systems: Clean 2 mg DOC/L 

PT 5   drinking water in reservoirs: origin raw water: Dirty 15 mg DOC/L; origin from drinking water supplier only: Clean 2 mg 

DOC/L 

   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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PT 5   drinking water for animals: origin raw water: Dirty 15 mg DOC/L origin from drinking water supplier only: Clean 2 mg 

DOC/L 

PT 5 

collective drinking water systems: Interfering substance 2 mg/L DOC (simulated use test) 

5  For  PT  01  or  PT02-'medical  applications'  the  required  tests  differ  from  PT01  or  PT02-'non-medical'  applications.  The  first  test  is  for 
medical applications and the second is for non-medical applications. In case both types of applications are claimed, only one test has to 
be carried out, in which the relevant worst case test conditions (in general medical test) are included. 

6 For hygienic handwash and handrub products used in hospitals the contact time is usually 30 seconds, for other uses the contact time 
is between 30 and 60 seconds (up to 120 seconds in case of virucidal activity). Please note that some EN tests were not developed for 
hand disinfection and therefore contact times shall be adapted. 
7 According to EN 1500 the test is passed when the mean reduction achieved by the hygienic handrub product under test is at least not 
inferior to that achieved by a reference handrub with propan-2-ol 60 % (v/v) (p=0.025). 
8 For handwash disinfectants it is assumed that hands will not be washed before washing with a disinfectant. Therefore, tests have to be 
done under dirty conditions. 

9  The  required  lg  reduction  in  EN  13727  /  EN  13624  is  lower  than  in  EN  1276  /  EN  1650,  as  in  EN  13727  and  EN  13624  for  hygienic 
handwash products the highest accepted concentration tested is 50%. 

10 According to EN 1499 the test is passed when the  mean reduction achieved by the hygienic handwash with the product under test is 
larger than that achieved by a specified reference hygienic handwash (unmedicated liquid soap) (p=0.01). 

11  The  WHO  states  that  for  several  products,  scrubbing  for  2-3  minutes  reduces  bacterial  counts  to  acceptable  levels.  However,  in  the 
past, longer scrubbing times were accepted. Contact times of longer than 3 minutes, and up to 5 minutes, will only be authorised with a 
sound justification on the necessity of such long scrubbing times. Shorter contact times are accepted when tested at this contact time. 
12 According to EN 12791 the test is passed when the mean reduction achieved by the surgical handrub product under test is at least not 
inferior to that achieved by a reference handrub with propan-1-ol 60 % (v/v). 
13  Products  intended  to  disinfect  surfaces  that  are  likely  to  come  into  contact  with  the  patient  and  /  or  the  medical  staff  and  surfaces, 
which  are  frequently  touched  by  different  people,  leading  to  the  transmission  of  microorganisms  to  the  patient,  must  be  tested  with  a 
contact time of maximum 5 min. The same applies where the contact time of the product must be limited for practical reasons.  Products 
for other surfaces than stated above may be tested with a contact time of maximum 60 min. 
14 When a surface disinfectant is a wipe soaked with disinfectant liquid the product should be tested in the EN16615 test (phase 2, step 
2), with mechanical action. 
15  For  PT  2:  as  a  phase  2,  step  2  an  EN  medical  area  test  with  adenovirus  and  murine  norovirus  may  be  used  (as  soon  as  available). 
Please note that although the virucidal claim for PT 2 includes testing against poliovirus, it is not required to test against this test species 

 
 
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in  the  phase  2,  step  2  test.  For  PT  4:  either  a  modified  EN  medical  test  or,  as  soon  as  available,  an  EN  food  area  test  with  Murine 
Norovirus. 
16 This test includes the use of milk as an interfering substance in order to maintain viability of the micro-organisms on the carriers during 
the test. Depending on the area of use, other suitable interfering substances should be tested (e.g. blood for use in hospitals). 
17 NF T 72-281 is a test from AFNOR, the French standardisation body. This standard is taken as a start to develop a new EN standard on 
airborne  disinfection  of  surfaces.  Where  available  an  EN  test  may  be  used  for  a  semi-field  method  which  evaluates  the  efficacy  of 
disinfectants when vaporised in a room. 

18 For medical equipment, tests with fungi and viruses are a basic requirement. For all other equipment, tests with fungi and viruses are 
optional. 

19  When  the  product  is  intended  to  be  used  at  high  temperatures  (>40  ºC)  relevant  test  organisms  for  these  temperatures  should  be 
used. See section 5.4.0.4.4 of this guidance (sub-section “Temperature”). 

20  The  interfering  substance  most  appropriate  for  the  in-use  conditions  should  be  used.  For  instance,  blood  for  products  used  in  the 
medical  area  and  protein  for  products  used  in  industry,  institutional  and  domestic  areas  are  recommended.  The  soiling  on  a  domestic 
product for use in pre-soak (dirty clothes) will be very much higher than the soiling present for a post-wash rinse additive (clean clothes). 
For  products  used  during  pre-soak  and  wash,  tests  should  be  done  under  dirty  conditions.  For  products  used  during  post-wash  rinse, 
tests  should  be  done  under  clean  conditions.  EN  16616:  The  obligatory  interfering  substance  to  be  tested  is  sterile  defibrinated  sheep 
blood (12.5 ml sheep blood per kg textile). 

21 EN 16616 should be used for biocidal products used in washing machines. For products not intended to be used in washing machines, 
small scale laboratory setting (e.g. for pre-soaking in a bucket) may be considered (e.g. ASTM E4206 or ASTM E2274). 

22 For surface disinfection in veterinary areas the normal contact time is 5 minutes. The maximum contact time is 30 minutes. For surface 
disinfection on animal transport vehicles the maximum contact time is 5 minutes. For disinfectants used on boots applied by spraying or 
walk-through bath the  contact time should not exceed 1 minute. 

23 As soon as a phase 2, step 2 test for porous surfaces are available these should be used (where relevant). 

24 For post-milking teat disinfection the normal contact time is 1 minute. The maximum contact time is 5 minutes.  For pre-milking teat 
disinfection the normal contact time is 10-30 seconds. The maximum contact time is 60 seconds. 

25 Presently there are no standard phase 2, step 2 tests for yeasts for teat disinfectants. As soon as a validated teat disinfectant phase 2, 
step 2 test is available for yeasts, this should be used. 

26 For disinfection in a hoof bath, information should be provided on how long the efficacy of a hoof bath can be guaranteed. This can be 
done in a challenge test or a field test. See the guidance chapter 5.4.3.4.2 for more details. 

   
 
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27 For hoof disinfection it is not anticipated that hoofs will be cleaned sufficiently before disinfection in practice. Therefore only tests under 
dirty conditions are acceptable. 

28 As long as no standard phase 2, step 2 tests are available it is not obligatory to provide 1 these tests. Phase 2, step 2 tests have to be 
provided as soon as standard tests are available. 

 29  For  uses  where  efficacy  against  bacteriophages  only  is  claimed,  EN  13610  can  be  employed.  For  all  other  PT4  uses  where  virucidal 
activity is claimed EN 14476 should be used with Norovirus and Adenovirus as test organisms. 

30 The test temperature should be according to the use instructions on the label: e.g. cold = 4, 10 ºC, no restriction = 20 ºC, high temp = 
40, to 80 ºC. When the product is intended to be used at high temperatures relevant test organisms for these temperatures should be 
used. See section 5.4.0.4.4 of this guidance (sub-section “Temperature”). 

31 UBA method “Quantitative determination of the efficacy of drinking water disinfectants”. This guidance for testing the bactericidal and 
virucidal activity in drinking water in a simulated use test is available at 
https://www.umweltbundesamt.de/sites/default/files/medien/374/dokumente/150629_version_2_-
_quantitative_determination_of_the_efficacy_of_drinking_water_disinfectants.pdf 

32 For continuous use the contact time is max 30 min. For reservoir water for animals the contact time should be as claimed. 

 
 
 
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Appendix 5. Examples  of  viruses  sorted  according  to 
their  presence  in  the  human  body  in  case  of  virus 
infection 

These viruses may contaminate hands, instruments, other surfaces and textiles. 

NOTE 1 This list is not exhaustive. 

NOTE 2 Enveloped viruses are in bold. 

Table 34: Examples of viruses 

Blood  

Enterovirus 

Filoviridae 

Flavivirus 

Hepatitis C virus (HCV) 

Hepatitis Delta virus (HDV) 

Human Immunodeficiency Virus (HIV) 

Herpesviridae 

Human T Cell Leukaemia Virus (HTLV) 

Hepatitis A Virus (HAV) 

Parvovirus B 19 

Hepatitis B virus (HBV) 

Respiratory tract 

Adenovirus (Mast-) 

Coronavirus 

Enterovirus 

Herpesviridae 

Neuronal tissue, ear,nose & eye 

Influenza Virus 

Paramyxoviridae 

Rhinovirus 

Rubella Virus 

Adenovirus (Mast-)  

Human Immunodeficiency Virus (HIV) 

Enterovirus 

Herpesviridae 

Measles Virus 

Gastro-intestinal 

Adenovirus(Mast-) 

Caliciviridae 

Coronavirus 

Astrovirus 

Polyomavirus 

Rabies Virus 

Rubella Virus 

Enterovirus 

Hepatitis A Virus (HAV) 

Hepatitis E Virus (HEV) 

Rotavirus 

Skin, breast and/or milk 

Enterovirus 

Herpesviridae 

Human Immunodeficiency Virus 
(HIV) 

Human T Cell Leukaemia Virus (HTLV) 

Papillomavirus 

Poxviridae 

   
 
 
 
 
 
 
 
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Spleen  and  lymph  nodes  (see  also 
blood) 

Human T Cell Leukaemia Virus 
(HTLV) 

Human Immunodeficiency Virus 
(HIV) 

Dental procedure 

Adenovirus(Mast-) 

Enterovirus 

Herpesviridae 

Hepatitis B virus (HBV) 

Urogenital tract 

Hepatitis C Virus (HCV) 

Hepatitis Delta Virus (HDV) 

Human Immunodeficiency Virus (HIV) 

Hepatitis B Virus (HBV) 

Human T Cell Leukaemia Virus (HTLV) 

Herpesviridae 

Human Immunodeficiency Virus 
(HIV) 

Papillomavirus 

Polyomavirus 

Reference: 

Van Regenmortel MHV et al.,Eds.: Virus Taxonomy, Classification and Nomenclature of 
Viruses, seventh report of the international committee on taxonomy of viruses. Academic 
Press, San Diego, 2000 

 
 
 
 
 
 
 
 
 
 
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Appendix 6.  Selection  of  recommended  tests  for  solid 
materials (excluding wood-preservatives)44 

Table 35: Selection of recommended tests for solid materials (excluding wood-
preservatives) 

Standard Method + 

Title 

Description 

section reference  

Possible 

application area 

ISO 22196, 

Measurement of 

Test to measure inhibition of 

Treated articles in PT 

Section 5.4.2.2 

antibacterial 

bacterial growth on plastic 

2, 3, 4, with a claim 

activity on 

material used in wet or humid 

to protect 

plastics and other 

conditions. 

non-porous 

surfaces 

people/animals by 

inhibition of bacterial 

growth. 

Section 5.4.2.3, Figure 

Simulated Splash 

Test to measure killing on 

Treated articles in PT 

4 

Model Non-

contact for non-porous material 

2, 3, 4, with a claim 

Porous Materials 

when the contaminant is spread 

to protect 

by splashes. Speed of required 

people/animals by 

effect (5-60 min) depends on 

killing on contact to 

claim. 

prevent cross-

contamination 

Section 5.4.2.3, Figure 

Simulated Splash 

Test to measure killing on 

Treated articles in PT 

5 

Model Porous 

contact for porous material 

2, 3, 4, with a claim 

Materials 

when the contaminant is spread 

to protect 

by splashes. Speed of required 

people/animals by 

effect (5-60 min) depends on 

killing on contact to 

claim. 

prevent cross-

contamination 

Section 5.4.2.3, Figure 

Printing Model 

Test to measure killing on 

Treated articles in PT 

6 

contact for non-porous material 

2, 3, 4, with a claim 

when the contaminant is spread 

to protect 

by e.g. hand-contact. Speed of 

people/animals by 

required effect (5-60 min) 

killing on contact to 

depends on claim. 

prevent cross-

contamination 

BS 3900 Part G6,  

Methods of test 

Painted panels inoculated with a 

PT 7 

Section 5.5.8.1 

for paints. Part 

mixture of spores of fungi 

G6: Assessment 

known to colonise paints 

of resistance to 

exposed to humid conditions for 

fungal growth 

up to 12 weeks should show 

visual appearance of fungal 

44 These tests are not necessarily appropriate for all claims and materials. Tests have to be chosen 
depending  on  the  claim  made,  the  materials  used  and  the  conditions  of  use  foreseen  for  the 
treated material/article. 

   
 
 
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381 

Standard Method + 

Title 

Description 

section reference  

Possible 

application area 

ASTM G21-09, 

Section 5.5.8.2 

growth. The treated sample 

should be free of it. 

Standard Practice 

The synthetic polymer portion of 

PT 7, 9 

for Determining 

plastic materials is usually 

Resistance of 

fungus-resistant in that it does 

Synthetic 

Polymeric 

not serve as a carbon source for 

the growth of fungi. It is 

Materials to Fungi 

generally the other components, 

such as plasticizers, cellulosics, 

lubricants, stabilizers, and 

colorants, that are responsible 

for fungus attack on plastic 

materials. 

ISO 846: 1997, 

Section 5.5.8.2 

Plastics - 

Method for determining the 

PT 7, 9 

Evaluation of the 

deterioration of plastics due to 

action of 

the action of fungi and soil 

microorganisms 

microorganisms by visual 

appearance, changes in mass or 

changes in physical properties. 

The aim is not to determine the 

biodegradability of plastics. 

Includes even a soil burial 

variant. 

Note: the section covering 

bacteria is not considered to be 

useful. 

ISO 16869:2008, 

Plastics - 

Method for determining the 

PT 7, 9 

Section 5.5.8.2 

Assessment of 

effectiveness of fungistatic 

the effectiveness 

compounds in protecting 

of fungistatic 

susceptible ingredients like 

compounds in 

plasticizers, stabilizers, etc., in 

plastics 

plastics formulations. A 

formulations 

minimum diffusion of the 

fungicide out of the matrix is 

necessary as the spores are 

added in an agar-layer. 

Evaluation by visual 

examination. 

BS EN 60068-2-

Environmental 

Test for fungal and microbial 

PT 7, 9 

10:2005, 

testing. Tests. 

resistance applicable to a wider 

Section 5.5.8.1 

Test J and 

range of materials 

guidance: Mold 

 
 
 
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Standard Method + 

Title 

Description 

section reference  

Possible 

application area 

growth 

OECD (OECD 

Guidance 

Method for measuring the 

Anti-odour testing for 

ENV/JM/MONO(2014)18 

Document for 

inhibition of bacterial growth or 

textiles, PT 9 

Section 5.5.8.5.2 

Quantitative 

metabolism of porous and non-

porous materials that have been 

treated with a biocide. 

Method for 

Evaluating 

Antibacterial 

Activity of Porous 

and Non-Porous 

Antibacterial 

Treated Materials. 

IBRG TEX13-005.4, 

Tier 1 Textile 

Method to determine the basic 

Anti-odour testing for 

Section 5.5.8.5.2 

Method 

antibacterial properties of 

textiles, PT 9 

Antibacterial 

textiles and porous materials 

Properties 

and articles treated with a 

biocide. 

   
 
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383 

Appendix 7.  Selection  of  recommended  tests  for  liquid 
materials45  

Table 36: Selection of recommended tests for liquid materials 

Reference + 

Title 

Description 

section reference 

Possible 

application area 

IBRG P 16-001.2, 

Tier 1 Wet State Paint 

A Method for Determining the 

PT 6 

Section 5.5.7 

Method 

Basic Efficacy of Biocidal Active 

Substances in aqueous based 

paints. 

IBRG PDG 16-001.2,  

Tier 1 Polymer 

A Method for Determining the 

PT 6 

Section 5.5.7 

dispersion Method 

Basic Efficacy of Biocidal Active 

Substances used in polymer 

dispersions. 

IBRG PDG 16-007.2, 

Tier 1 Basic Efficacy 

Method for determining the 

PT 6 

Section 5.5.7 

Method for Biocidal 

basic efficacy of biocidal active 

Active Substances 

substances for in-can 

used to Preserve 

preservation in aqueous based 

Aqueous-Based 

products 

Products  

IBRG FFG 16-001.4, 

Tier 1 Metal Working 

Method for determining the 

PT 13 

Section 5.5.13 

Fluids Method 

basic efficacy of biocidal active 

substances in aqueous based 

metalworking fluids. 

45 These tests are not necessarily appropriate for all claims and materials. Tests have to be chosen 
depending  on  the  claim  made,  the  materials  used  and  the  conditions  of  use  foreseen  for  the 
treated material/article. 

 
 
 
 
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Appendix 8. Commonly  Used  Methods  to  Measure  the 
in  Liquid 
Effects  of  Preservative/Curative  Action 
Matrices46 

Table 37: Commonly Used Methods to Measure the Effects of 
Preservative/Curative Action in Liquid Matrices 

Reference  Title 

Description 

ASTM 
D2574-06 

Standard Test Method for Resistance 
of Emulsion Paints in the Container 
to Attack by Microorganisms  

ASTM 
D4783-
01e1 

Standard Test Methods for 
Resistance of Adhesive Preparations 
in Container to Attack by Bacteria, 
Yeast, and Fungi 

ASTM 
E1259-05 

Standard Practice for Evaluation of 
Antimicrobials in Liquid Fuels Boiling 
Below 390°C 

This test method covers the determination 
of the relative resistance of emulsion 
paints to attack in the container by 
microorganisms. 

Determination of the resistance of liquid 
adhesive preparations to microbial attack 
in the container by challenging adhesive 
specimens with cultures of bacteria, yeast, 
or fungi, and checking for their ability to 
return to sterility. These test methods 
return qualitative results. 

The procedure should be used to evaluate 
the relative efficacy of microbicides in 
liquid fuels boiling below 390°C. The effect 
of environmental conditions, such as a 
variety of fuel additives, metal surfaces, 
and climatology, are variables that can be 
included in specific tests using this 
protocol. 

SABS 1102 
(1987) 

Bacterial efficacy of biocides used in 
water-based emulsion paints 

Efficacy test for in can preservatives in 
paints (emulsion) against bacteria.  

NF X41-
520 March 
1968 

Protection. Testing method for 
resistance of paints to 
microorganisms and their protective 
power. 

Standard Practice for Evaluating 
Water-Miscible Metalworking Fluid. 
Bioresistance and Antimicrobial 
Pesticide Performance 

ASTM 
E2275-
03e1 
(replaces 
D3946 and 
E686) 

Laboratory procedures for rating the 
relative inherent bioresistance of water-
miscible metalworking fluids, the 
bioresistance attributable to augmentation 
with antimicrobial pesticides or both, for 
determining the need for microbicide 
addition prior to or during fluid use in 
metalworking systems and for evaluating 
microbicide performance.  
Relative bioresistance is determined by 
challenging metalworking fluids with a 
biological inoculum that may either be 
characterized (comprised of one or more 
known biological cultures) or 

PT 

6 

6 

6  

6 

6 

13 

46 Please note: The methods listed are not necessarily appropriate in all cases. Their applicability 
depends  on  the  claim  made,  the  materials  used  and  the  conditions  of  use  for  the  treated 
material/article. These methods are listed to give an overview for the assessor when and where a 
method is meaningful to demonstrate a claim and where its limits are. 

   
 
 
 
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Reference  Title 

Description 

385 

PT 

uncharacterized (comprised of biologically 
contaminated metalworking fluid or one or 
more unidentified isolates from 
deteriorated metalworking fluid). 
Challenged fluid bioresistance is defined in 
terms of resistance to biomass increase, 
viable cell recovery increase, chemical 
property change, physical property change 
or some combination thereof. 
This practice is applicable to antimicrobial 
agents that are incorporated into either the 
metalworking fluid concentrate or end-use 
dilution. It is also applicable to 
metalworking fluids that are formulated 
using non-microbicidal, inherently 
bioresistant components. 
The results of tests completed in 
accordance with this practice should be 
used only to compare the relative 
performance of products or microbicide 
treatments included in a test series. 
Results should not be construed as 
predicting actual field performance. 

This laboratory test method is designed to evaluate the 
utility and effectiveness of antimicrobial agents intended 
to control microbial growth in invert emulsions and other 
water containing hydraulic fluids. 

13 

Determines the relative bioresistance of 
aqueous metalworking fluids towards non-
tuberculous (NTM), rapidly growing (RGM), 
environmental mycobacteria by 
challenging them with a mycobacterial 
inoculum isolated from actual spoiled 
metalworking fluid field samples from the 
user/s site. 
In order to simulate field conditions, 
another challenge inoculum consisting of a 
mixture of common metalworking fluid 
spoilage microorganisms originating from 
actual MWF field samples is also used 

13 

13 

13 

13 

ASTM 
E979-
91(2004) 

ASTM 
WK8252 

Standard Test Method for Evaluation 
of Antimicrobial Agents as 
Preservatives for Invert Emulsion 
and Other Water Containing 
Hydraulic Fluids 

New Standard Test Method for 
Determining Resistance of Aqueous 
Metalworking Fluids towards Non-
Tuberculous, Environmental 
Mycobacteria 

SABS 
1435-1987  

South African standard specification 
for biocides for use in emulsions of 
aqueous metal working fluid and 
aqueous hydraulic fluid.  

Rawlinson 
and 
Shennan, 
1987.  

A recirculating test rig for the 
investigation of metal-working fluid 
spoilage. In Industrial 
microbiological testing 1987 pp. 
227-231. Edited by Hopton and, 
J.W.; Hill, E.C. 

The method described, which attempts to 
simulate the conditions under which a 
metal working fluid will be used in service, 
has been used extensively for the testing 
of new product formulations and the 
evaluation of biocides. 

Cutting fluid, soluble, biostable joint 
service designation ZX-9  

UK MOD 
91-70 
issue 
(1990)  

 
 
 
 
 
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Appendix 9. Commonly  Used  Methods  to  Measure  the 
Effects of Protecting Material47 

Table I: Methods used to Examine the Resistance of Porous Materials to 
Biodeterioration: Textiles 

Reference 

Title 

Description 

EN 
14119:2003 

Testing of textiles –
Evaluation of the 
action of microfungi 

The test is designed to determine the 
susceptibility of textiles to fungal growth. 
Assessment is by visual rating and 
measurement of tensile strength. 

Major 
Principle/Use 

Agar plate test 

Agar plate test 

The two purposes of the test are to determine 
the susceptibility of textiles to microfungi and 
to evaluate the efficacy of fungicides on 
textiles. 

AATCC 30-
2004 

DIN 53931 

MIL-STD-
810F 

BS 6085 
:1992 

EN ISO 
11721-1 
(2001) 

Antifungal activity, 
Assessment on 
textile materials: 
mildew and rot 
resistance of textile 
materials 

Testing of textiles; 
determination of 
resistance of textiles 
to mildew; growth 
test 

Environmental 
Engineering 
considerations and 
laboratory tests;  
Method 508.5 
FUNGUS 

Determination of the 
resistance of textiles 
to microbial 
deterioration 

Textiles -  
Determination of 
resistance of 
cellulose-containing 
textiles to micro-
organisms: Soil 
burial test 
Part 1: Assessment 
of rot retarding 
finishing 

The test determines the efficacy of treatments 
for prevention of fungal growth on/in textiles. It 
also allows the performance testing of a 
treatment after UV irradiation , leaching etc. 

Agar plate test 

The purpose of the method is to assess the 
extent to which a material will support fungal 
growth and how performance of that material is 
affected by such growth. 

Humid chamber 
test (90 to 99% 
humidity) 

The purpose of the method is to assess the 
extent to which a material will support 
fungal/bacterial growth and how performance 
of the material is affected by such growth. 
Visual Assessment and measurement of tensile 
strength. 

a) soil burial 
test; 
b) agar plate 
test, 
c) humid 
chamber test 

The test is designed to determine the 
susceptibility of cellulose containing textiles 
against deterioration by soil micro-organisms. 
 Preserved and unpreserved textiles are 
compared. Visual Assessment and 
measurement of tensile strength. 

Soil burial test  

47 Please note: The methods listed are not necessarily appropriate in all cases. Their applicability 
depends  on  the  claim  made,  the  materials  used  and  the  conditions  of  use  for  the  treated 
material/article. These methods are listed to give an overview for the assessor when and where a 
method is meaningful to demonstrate a claim and where its limits are. 

   
 
 
 
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Reference 

Title 

Description 

EN ISO 
11721-2 
(2003) 

BS 2011 : 
Part 2.1J 
(IEC 68-2-
10) 

AS 1157.2 - 
1999 

AS 1157.4 - 
1999 

AS 1157.3 - 
1999 

387 

Major 
Principle/Use 

Soil burial test 

Humid chamber 
test (90 to 99% 
humidity) 

Agar plate test 

Agar plate test 

The test identifies the long-term resistance of a 
rot-retardant finish against the attack of soil 
inhabiting micro-organisms. It allows to make a 
distinction between regular long-term 
resistance and increased long-term resistance. 
Visual Assessment and measurement of tensile 
strength 

Textiles -  
Determination of 
resistance of 
cellulose-containing 
textiles to micro-
organisms: Soil 
burial test 
Part 2: Identification 
of long-term 
resistance of a rot 
retardant finish 

Basic environmental 
testing procedures 

Mould growth test to show the susceptibility of 
a material towards colonization by fungi. 

Australian Standard - 
Methods of Testing 
Materials for 
Resistance to Fungal 
Growth 
Part 2: Resistance of 
Textiles to Fungal 
Growth.  Section 1 - 
Resistance to 
Surface Mould 
Growth. 

Australian Standard - 
Methods of Testing 
Materials for 
Resistance to Fungal 
Growth 
Part 2: Resistance of 
Textiles to Fungal 
Growth.  Section 2 - 
Resistance to 
Cellulolytic Fungi. 

Australian Standard - 
Methods of Testing 
Materials for 
Resistance to Fungal 
Growth 
Part 2: Resistance of 
Cordage and Yarns 
to Fungal Growth. 

Test specimens are inoculated with a 
suspension of spores of Aspergillus niger and 
then incubated on the surface of a mineral salts 
based agar for 14 days and then assessed for 
growth.  Both leached and unleached 
specimens are examined. Glass rings are 
employed to hold the specimens in intimate 
contact with agar when necessary. Specimens 
are examined for the presence of surface mould 
growth. 

Test specimens are inoculated with a 
suspension of spores of Chaetomium globosum 
and then incubated on the surface of a mineral 
salts based agar for 14 days and then assessed 
for growth. Both leached and unleached 
specimens are examined and exposed samples 
are subjected to a tensile strength test.  Glass 
rings are employed to hold the specimens in 
intimate contact with agar when necessary. 

Test specimens are inoculated with a 
suspension of spores of Chaetomium globosum 
and then incubated on the surface of a mineral 
salts based agar for 14 days and then assessed 
for growth.  Both leached and unleached 
specimens are examined and exposed samples 
are subjected to a tensile strength test. 

Agar plate test 
(other vessels 
containing 
media are 
employed for 
large 
specimens). 

 
 
 
 
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Table II: Methods used to Examine the Resistance to Biodeterioration: Geotextile 

Reference 

Title 

Description 

EN 
12225:2000 

Geotextiles and 
Geotextiles-related 
products - Method 
for determining the 
microbiological 
resistance by a soil 
burial test 

The test is designed to determine the 
susceptibility of geotextiles and related 
products to deterioration by soil micro-
organisms. Visual Assessment and 
measurement of tensile strength. 

Major Principle 

Soil burial test 

Table III: Methods used to Examine the Antimicrobial Activity and Microbial 
Resistance of Paper etc. 

Reference 

Title 

Description 

DIN EN 1104 
- 05  

ASTM D 
2020-03  

ASTM D 
2020-03  

AS 1157.7 - 
1999 

AS 1157.5 - 
1999 

Paper and board 
intended to come 
into contact with 
foodstuffs 
Determination of 
transfer of 
antimicrobic 
constituents 

Standard Test 
Methods for Mildew 
(Fungus) Resistance 
of Paper and 
Paperboard - Direct 
Inoculation 

Standard Test 
Methods for Mildew 
(Fungus) Resistance 
of Paper and 
Paperboard - Soil 
Burial 

Australian Standard - 
Methods of Testing 
Materials for 
Resistance to Fungal 
Growth 
Part 6: Resistance of 
Papers and Paper 
Products to Fungal 
Growth. 

Australian Standard - 
Methods of Testing 
Materials for 
Resistance to Fungal 
Growth 
Part 5: Resistance of 
Timber to Fungal 
Growth. 

A minimum of 20 replicate sub-samples (each 
10 - 15 mm in diameter) taken from 10 
samples of a batch of paper are placed in 
intimate contact with nutrient agar plates 
inoculated with either Bacillus subtilis or 
Aspergillus niger and incubated at 30° C for 7 
days and at 25° C for 8 - 10 days respectively. 

Major Principle 

Zone Diffusion 
Assay. 

Replicate samples (3) are inoculated with a 
suspension of fungal spores and then incubated 
on the surface of a minimal mineral-salts 
medium to determine if they support fungal 
growth. 

Biodeterioration 
Test. 

Replicate samples (5) are buried in soil for 14 
days and then examined for the deterioration 
compared with unburied samples for both 
physical deterioration and loss of tensile 
strength. 

Biodeterioration/
Biodegredadatio
n Test. 

Test specimens are placed on the surface of a 
mineral-salts based agar and then both the 
specimen and the agar are inoculated with a 
suspension of spores of a range of fungi. They 
are then incubated for 14 days and then 
assessed for growth. Growth on the specimen is 
assessed. 

Agar plate test 

Test specimens are placed on the surface of a 
mineral salts based agar and then both the 
specimen and the agar are inoculated with a 
suspension of spores of a range of fungi. They 
are then incubated for 14 days and then 
assessed for growth. Growth on the specimen is 
assessed. 

Agar plate test 

   
 
 
 
 
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Reference 

Title 

Description 

AS 1157.6 - 
1999 

Australian Standard - 
Methods of Testing 
Materials for 
Resistance to Fungal 
Growth 
Part 6: Resistance of 
Leather and Wet 
‘Blue’ Hides to 
Fungal Growth. 

Test specimens are placed on the surface of a 
mineral salts based agar and then both the 
specimen and the agar are inoculated with a 
suspension of spores of a range of fungi. They 
are then incubated for 14 days and then 
assessed for growth. Both leached and 
unleached specimens are examined. Growth on 
specimens is assessed. Sucrose containing 
media is employed where true controls cannot 
be obtained. 

389 

Major Principle 

Agar plate test 

Table IV: Methods used to Examine the Resistance to Biodeterioration: Plastics 

Reference 

Title 

Description 

Humid chamber test (90 to 99% humidity) 

Major Principle 

Biodegradability 
test 

Humid chamber test (90 to 99% humidity) 

Agar plate test 

ASTM D 
5338 - 92 

Humid chamber test 
(90 to 99% 
humidity) 

ASTM E 1428 
- 99 

Humid chamber test 
(90 to 99% 
humidity) 

ASTM G 22 - 
76 

ASTM G 21 - 
96 

ASTM G 29 - 
96 

EN 
14047:2002 

EN 
14048:2002 

ISO 
846:1997 

EUROCAE 
ED-14B/ 
RTCA DO 
160B 

MIL-STD-
810F 

BS 2011 : 
Part 2.1J 
(identical 

Agar plate test 

Agar plate test 

Agar plate test 

Agar plate test 

Agar plate test 

Agar plate test 

Agar plate test 

Agar plate test 

Humid chamber test 
(90 to 99% 
humidity) 

Humid chamber test 
(90 to 99% 
humidity) 

Humid chamber test (90 to 99% humidity) 

Humid chamber test (90 to 99% humidity) 

Agar plate test 

Agar plate test 

Agar plate test 

Agar plate test 

Biofouling test 

Biodegradability 
test 

Biodegradability 
test 

Agar plate test; 
soil burial test 

Humid chamber 
test ( 90 to 99% 
humidity) 

Environmental 
Engineering 
considerations and 
laboratory tests;  
Method 508.5 
FUNGUS 

The purpose of the method is to assess the 
extent to which a material will support fungal 
growth and how performance of the material is 
affected by such growth. 

Humid chamber 
test (90 to 99% 
humidity) 

Basic environmental 
testing procedures 

Mould growth test to show the susceptibility of 
a material towards the colonization by fungi. 

Humid chamber 
test (90 to 99% 
humidity) 

 
 
 
 
 
 
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Reference 

Title 

Description 

Major Principle 

with IEC 68-
2-10) 

ISO 
16869:2008 

AS 1157.4 - 
1999 

AS 1157.11 - 
1999 

AS 1157.11 - 
1999 

Plastics - 
Assessment of the 
effectiveness of 
fungistatic 
compounds in 
plastics formulations 

A specimen is placed on a nutrient-salt- agar 
(without additional carbon source) in a petri 
dish and overlayed with the same agar 
containing fungal spores. Rate of growth on the 
specimen is visually assessed. 

Australian Standard 
- Methods of Testing 
Materials for 
Resistance to Fungal 
Growth 
Part 4: Resistance of 
Coated Fabrics and 
Electronic Boards to 
Fungal Growth. 

Test specimens are inoculated with a 
suspension of spores of Chaetomium globosum 
and then incubated on the surface of a mineral 
salts based agar for 14 days and then assessed 
for growth. Both leached and unleached 
specimens are examined and exposed samples 
are subjected to a tensile strength test. Glass 
rings are employed to hold the specimens in 
intimate contact with agar when necessary. 

Agar plate test 

Agar plate test 

Australian Standard 
- Methods of Testing 
Materials for 
Resistance to Fungal 
Growth 
Part 11: Resistance 
of Rubbers and 
Plastics to Surface 
Fungal Growth - 
Section 1: 
Resistance to Growth 

Australian Standard 
- Methods of Testing 
Materials for 
Resistance to Fungal 
Growth 
Part 11: Resistance 
of Rubbers and 
Plastics to Surface 
Fungal Growth - 
Section 2: 
Fungistatic 
Properties 

Agar plate test 

Test specimens are inoculated with a 
suspension of spores of a range of fungi and 
then incubated on the surface of a mineral salts 
based agar for 14 days and then assessed for 
growth. Both leached and unleached specimens 
are examined. Glass rings are employed to hold 
the specimens in intimate contact with agar 
when necessary. 

Agar plate test 

Test specimens are placed on the surface of a 
sucrose, mineral salts based agar and then both 
the specimen and the agar are inoculated with 
a suspension of spores of a range of fungi.  
They are then incubated for 14 days and then 
assessed for growth. Both leached and 
unleached specimens are examined. Glass rings 
are employed to hold the specimens in intimate 
contact with agar when necessary. Growth on 
both the specimen and inhibition of growth on 
the surrounding agar are assessed. 

Table V: Methods used to Examine the Antimicrobial Activity and Microbial 
Resistance of Surface Coatings & Adhesives 

Reference 

Title 

Description 

BS3900 Part 
G6 

Assessment of 
resistance to fungal 
growth 

Replicate test panels coated with the test 
coating are inoculate with a suspension of 
spores of fungi known to grow on the surface of 
paints and related materials.  The samples are 
then incubated under conditions suitable to 
support fungal growth (23 ± 2°C and high 
humidity/surface condensation). In the 

Major 
Principle 

Biodeterioration 
Test 

   
 
 
 
391 

Major 
Principle 

Biodeterioration 
Test 

published standard, condensation on the test 
panels is achieved by increasing the 
temperature in a water bath below the samples 
for short periods of time.  Revisions are in 
progress which may obviate this step. The 
method is validated if fungal 
growth/germination of spores is observed after 
two weeks on a standard coating known to be 
susceptible to fungal growth. After incubation 
growth is rated in accordance with a scale 
related to the percent cover with fungal growth 
(following visual and microscopical 
examination). A natural and artificial soiling are 
described in the method which can be 
employed when appropriate. 

Replicate test panels coated with the test 
coating are inoculated with a suspension of 
spores of fungi known to grow on the surface of 
paints and related materials.  The samples are 
then incubated under conditions suitable to 
support fungal growth. 

Replicate test panels coated with the test 
coating are inoculated with a suspension of 
spores of fungi known to grow on the surface of 
paints and related materials.  The samples are 
then incubated under conditions suitable to 
support fungal growth. 

Biodeterioration 
Test 

Agar Plate Test 

Guidance on the BPR: Volume II Parts B+C  
Version 4.0 December 2021 

Reference 

Title 

Description 

ASTM 
D3273-12 

WK4201 

ASTM 
D5590-94 

Standard Test 
Method for 
Resistance to Growth 
of Mold on the 
Surface of Interior 
Coatings in an 
Environmental 
Chamber 

Standard Test 
Method for 
Resistance to Mold 
Growth on Building 
Products in an 
Environmental 
Chamber 

Standard Test 
Method for 
Determining the 
Resistance of Paint 
Films and Related 
Coatings to Fungal 
Defacement by 
Accelerated 
Four-Week Agar Plate 
Assay 

SS345 
Appendix 9 

Formal Title Missing 
at Present 

EN 
15457:2007 

Paints and varnishes 
– Laboratory method 
for testing the 
efficacy of film 
preservatives in a 
coating against fungi 

Biodeterioration 
Test. 

Zone Diffusion 
Assay 

The bottom of glass petri dishes are coated 
with paint. After drying, a culture of algae in a 
suitable growth liquid medium is placed into the 
dish and incubated under conditions suitable for 
algal growth. 

Coatings are applied to glass fibre discs and 
then placed in intimate contact with the surface 
of nutrient agar plates.  The coatings and 
surrounding media are then inoculated with a 
mixed suspension of spores of 4 fungal species 
selected from a list of 10. The plates are then 
incubated at 24°C for 21 days and then 
assessed for growth using a rating scale.  The 
test is intended to support claims that a biocide 

 
 
 
 
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Reference 

Title 

Description 

Major 
Principle 

AS 1157.10 
- 1999 

EN 
15458:2007 

Australian Standard - 
Methods of Testing 
Materials for 
Resistance to Fungal 
Growth 
Part 10: Resistance 
of Dried or Cured 
Adhesives to Fungal 
Growth 

Paints and varnishes 
– Laboratory method 
for testing the 
efficacy of film 
preservatives in a 
coating against algae 

VdL RL06 

Guideline to Evaluate 
the Resistance of 
Coating Materials 
against Mold Growth 

VdL RL07 

Guideline to Evaluate 
the Resistance of 
Coating Materials 
against Mold Growth 

can have an effect in a surface coating in 
support of its listing in the relevant use 
category within the EU BPD.  It is not intended 
to assess the performance of surface coatings. 

Test materials coated onto glass microscope 
slides are inoculated with a suspension of 
spores of a range of fungal species and then 
incubated on the surface of a mineral salts 
based agar for 14 days and then assessed for 
growth. 

Coatings are applied to glass fibre discs and 
then placed in intimate contact with the surface 
of nutrient agar plates. The coatings and 
surrounding media are then inoculated with a 
mixed suspension of 3 algal species selected 
from a list of 5. The plates are then incubated 
at 23°C under illumination (16 hour day length, 
1000 Lux) for 35 days and then assessed for 
growth using a rating scale.  The test is 
intended to support claims that a biocide can 
have an effect in a surface coating in support of 
its listing in the relevant use category within 
the EU BPD. It is not intended to assess the 
performance of surface coatings. 

Coatings are applied to paper discs and then 
placed in intimate contact with the surface of 
nutrient agar plates. The coatings and 
surrounding media are then inoculated with a 
mixed suspension of spores of A niger and 
Penicillium funiculosum. The plates are then 
incubated at 28°C for 3 weeks and assessed for 
growth using a rating scale after 1, 2 and 3 
weeks.  Coatings for exterior use and ‘wet’ 
applications are leached in water prior to 
testing. 

Coatings are applied to paper discs and then 
placed in intimate contact with the surface of 
nutrient agar plates.  The coatings and 
surrounding media are then inoculated with a 
mixed suspension of Scenedesmus vacuolaris 
and Stichococcus bacillaris. The plates are then 
incubated at 23°C for 3 weeks under 
illumination (16 hour day length, 1000 Lux) 
and assessed for growth using a rating scale 
after 1, 2 and 3 weeks. Coatings for exterior 
use and ‘wet’ applications are leached in water 
prior to testing. 

Agar plate test 

Zone Diffusion 
Assay 

Zone Diffusion 
Assay/Humid 
Chamber Test 

Zone Diffusion 
Assay/Humid 
Chamber Test 

   
 
 
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Table VI: Methods used to Examine the Antimicrobial Activity of Textiles 
(fabric, yarn or pile/wadding) 

Reference 

Title 

Description 

Major Principle 

JIS L 1902: 
2008 

Testing Method for 
Antibacterial Activity 
of Textiles 
Qualitative Test 

JIS L 1902: 
2008 

Testing Method for 
Antibacterial Activity 
of Textiles 
Quantitative Test 

EN ISO 
20645 - 
2004 

Textile Fabrics - 
Determination of the 
antibacterial activity 
- Agar plate test 
(ISO/FDIS 
20645:2004) 

Zone diffusion assay. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Cell suspension intimate 
contact test. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Zone diffusion assay. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Three replicate samples of 
fabric, yarn or pile/wadding are 
placed in intimate contact with 
the surface of agar plates that 
have been inoculated with a cell 
suspension of either 
Staphylococcus aureus or 
Klebsiella pneumoniae and 
incubated at 37° C for 24 - 48 
hours. The presence of and size 
of any zone of inhibition around 
the samples is then recorded. 

Replicate samples of fabric (6 of 
the control and 3 of the treated) 
are inoculated with individual 
bacterial species (e.g. S. aureus 
and K. pneumoniae) suspended 
in a heavily diluted nutrient 
medium. The samples are 
incubated under humid 
conditions at 37° C for a 
specified contact time. Activity is 
assessed by comparing the size 
of the initial population in the 
control with that present 
following incubation. No 
neutraliser is employed during 
cell recovery. 

Four replicate samples of fabric 
(25 ± 5 mm) are placed in 
intimate contact with a solid 
nutrient medium in a petri dish. 
The samples are then overlaid 
with molten solid nutrient media 
which has been inoculated with 
a cell suspension of either S. 
aureus, Escherichia coli or K. 
pneumoniae. The plates are 
then incubated for between 18 
and 24 hours and the plates are 
then assessed for growth based 
on either the presence of a zone 
of inhibition of  > 1 mm or the 
absence/strength of the growth 
in the media overlaying the test 
specimen. 

SN 195920 

Examination of the 
Antibacterial Effect 
of Impregnated 
Textiles by the Agar 
Diffusion Method 

Four replicate samples of fabric 
(25 ± 5 mm) are placed in 
intimate contact with a solid 
nutrient medium in a petri dish. 
The samples are then overlaid 
with molten solid nutrient media 

Zone diffusion assay. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

 
 
 
 
 
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Reference 

Title 

Description 

Major Principle 

SN195924 

Textile Fabrics - 
Determination of the 
Antibacterial 
Activity: 
Colony Plate Count 
Method 

SN195921 

Textile Fabrics - 
Determination of 
Antimycotic Activity: 
Agar Diffusion Plate 
Test 

Cell suspension intimate 
contact test. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Zone diffusion assay. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

which has been inoculated with 
a cell suspension of either S. 
aureus or E. coli. The plates are 
then incubated for between 18 
and 24 hours and the plates are 
then assessed as described in 
BS EN ISO 20645 above. 

Fifteen replicate samples (each 
replicate is comprised of 
sufficient specimens of 25 ± 5 
mm to absorb 1 ml of test 
inoculum) are inoculated with 
cells of either E. coli or S. 
aureus suspended in a liquid 
nutrient medium and incubated 
in sealed bottles for up to 24 
hours at 27° C. After 0, 6 and 
24 hours, 5 replicate samples 
are analysed for the size of the 
viable population present. A 
neutraliser is employed. An 
increase of 2 orders of 
magnitude of the population 
exposed to a control sample is 
required to validate the test.  
The method defines a textile as 
antibacterial if no more than a 
specified minimum level of 
growth is observed after 24 
hours in 4 of the 5 replicate 
groups of samples. 

Replicate (4) samples of 
sterilised fabric (25 ± 5 mm 
diameter) are placed in intimate 
contact with a solid nutrient 
medium in a petri dish. Each 
petri dish has been prepared as 
a double layer. The first layer 
consists of 10 ml nutrient agar, 
the second layer of another 10 
ml of the same nutrient agar to 
which 0.1 ml spore suspension 
(107 ml-1) of either Candida 
albicans, Aspergillus niger, 
Cladosporium sphaerospermum 
or Trichophyton mentagrophytes 
had been added. The plates are 
then incubated at 28° C either 2 
days (C. albicans) or 7 days ( A. 
niger, C. sphaerospermum and 
T. mentagrophytes). The test is 
valid when control specimens of 
the same material without 
biocide, or of a biocide-free 
standard specified cotton 
material are fully overgrown. 
Good antimycotic efficacy is 
considered to be demonstrated 

   
 
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395 

Reference 

Title 

Description 

Major Principle 

ISO 20743 

Textiles - 
Determination of 
antibacterial activity 
of antibacterial 
finished products: 
Absorption method 

ISO 20743 

Textiles - 
Determination of 
antibacterial activity 
of antibacterial 
finished products: 
Transfer method 

ISO 20743 

Textiles - 
Determination of 
antibacterial activity 
of antibacterial 
finished products: 
Printing method 

when the specimens show no 
fungal growth on their surface. 
The test specifies that both sides 
of a material have to be tested. 

Replicate (6) samples of textile 
are inoculated with a 
standardised broth culture of 
either S. aureus or K. 
pneumoniae in individual tubes 
and then incubated at 37° C for 
18 - 24 hours in closed 
containers. Samples are 
analysed for the presence of 
viable bacteria both before and 
after incubation by either total 
viable count or the 
determination of total ATP.  
Samples are sterilised prior to 
testing and a neutraliser is 
employed during recovery.  The 
test is validated by growth of ^1 
order of magnitude during the 
incubation period. 

Replicate (6) samples of test 
material are placed in contact 
with an agar plate that has been 
inoculated with a specified 
volume of a known cell 
suspension of either S. aureus 
and K. pneumoniae using a 200 
g weight for 1 minute. The 
samples are then removed. 
Replicate (3) samples are 
analysed for either the number 
of viable bacteria or the total 
ATM content both before and 
after incubation under humid 
conditions at 37° C for 24 hours. 
Samples are sterilised prior to 
testing and a neutraliser is 
employed during cell recovery. 
The test is validated by either 
growth of ^1 order of magnitude 
during the incubation period or 
by a measure of the variability 
of the data obtained. 

Replicate (6) samples of test 
material are either S. aureus 
and K. pneumoniae by ‘printing’ 
cells collected on a membrane 
filter onto their surface in a 
standardised manner. The 
samples are then incubated 
under humid conditions for 18 - 
24 hours at 20° C for a specified 
contact time(s). Replicate (3) 
samples are analysed for either 

Cell suspension intimate 
contact test. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Cell suspension intimate 
contact test. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

‘Dry’ inoculum intimate 
contact test. 
The transfer method of 
inoculation could be 
adapted to provide some 
simulation data. 

 
 
 
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Reference 

Title 

Description 

Major Principle 

ISO/FDIS 
13629-1 

Textiles - 
Determination of 
Antifungal Activity of 
Textile Products: 
Part 1 - 
Luminescence 
Method 

ISO/WD 
13629-1 

Textiles - 
Determination of 
Antifungal Activity of 
Textile Products: 
Part 2 - Plate Count 
Method 

the number of viable bacteria or 
the total ATM content both 
before and after incubation. 
Samples are sterilised prior to 
testing and a neutraliser is 
employed during cell recovery. 
The test is validated by either 
determining the survival of the 
inoculum on the control 
material. 

Samples of textiles are 
inoculated with a suspension of 
fungal spores either by direct 
application or transfer from an 
agar surface and then 
incubated. Germination and 
growth of the spores is followed 
by measuring the ATP 
concentration associated with 
the samples. The presence of an 
antifungal treatment is expected 
to show either an inhibition of 
germination or a reduction in the 
rate of growth as indicated by 
reduced concentrations of ATP 
associated with the treated 
material in comparison with the 
untreated material. 

Samples of textiles are 
inoculated with a suspension of 
fungal spores either by direct 
application or transfer from an 
agar surface and then 
incubated. Germination and 
growth of the spores is followed 
by measuring the number of 
colony forming units. The 
presence of an antifungal 
treatment is expected to show 
either an inhibition of 
germination or a reduction in the 
rate of growth as indicated by 
reduced numbers of colony 
forming units associated with 
the treated material in 
comparison with the untreated 
material. 

Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 
The transfer method of 
inoculation could be 
adapted to provide some 
simulation data. 

Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 
The transfer method of 
inoculation could be 
adapted to provide some 
simulation data. 

   
 
 
 
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Table VII: Methods used to Examine the Antimicrobial Activity of Carpets 

Reference 

Title 

Description 

Major Principle 

AATCC 
174-2011 

Antimicrobial Activity 
Assessment of 
Carpets 
Qualitative 
Antibacterial Activity 

AATCC 
174-2011 

Antimicrobial Activity 
Assessment of 
Carpets 
Quantitative 
Antibacterial Activity 

AATCC 
174-2011 

Antimicrobial Activity 
Assessment of 
Carpets 
Quantitative 
Antifungal Activity 

Petri dishes with nutrient media 
are inoculated with a single, 
diagonal streak (approx.7.5 cm) 
of either S. aureus or K. 
pneumoniae. An unsterilized test 
specimen (25 mm x 50 mm) is 
placed in intimate contact and 
transversely across the inoculum 
on the agar surface. The plates 
are then inoculated at 37° C for 
18 - 24 hours. The front and 
back of the carpet are tested 
separately. After incubation, the 
plates are inspected for the 
presence of growth both below 
the specimens and for any zone 
of inhibition surrounding the 
specimens. The test can also be 
used to test the effect of 
cleaning regimes. An untreated 
control is optional. 

Unsterilized specimens of carpet 
are pre-wetted with either 
sterile water or a wetting agent 
before being inoculated with 
individual suspensions of either 
S. aureus or K. pneumoniae in 
either a low or a high nutrient 
solution. The samples are then 
incubated in a tightly closed jar 
at 37° C for a specified contact 
time. Cells are recovered in 100 
ml of a neutraliser after 0 and 6 
- 24 hours of incubation. Activity 
is assessed by comparing the 
size of the initial population in 
the control (if used) with that 
present following incubation. A 
control is optional. When not 
employed, viable counts 
following incubation of the 
treated specimens alone are 
considered. The test can also be 
used to test the effect of 
cleaning regimes. 

Petri dishes containing 
Sabouraud Dextrose Agar are 
inoculated with 1 ml of a spore 
suspension of Aspergillus niger. 
Immediately afterwards, 
specimens (38 mm diameter) of 
unsterile test material are placed 
into intimate contact with the 
agar. An additional 0.2 ml of the 
same spore suspension is also 

Qualitative assessment of 
rate of kill and zone 
diffusion test 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Cell suspension intimate 
contact test. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Zone diffusion 
test/surface growth test. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

 
 
 
 
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Reference 

Title 

Description 

Major Principle 

WIRA Test F  Test Method for 

Assessing the 
Survival of Test 
Organisms on Floor 
Coverings 

employed to inoculate the test 
pieces directly. The samples are 
then incubated at 28°C for 7 
days.  The back and front of the 
discs of carpet are tested in 
separate dishes. The zone of 
inhibition and the growth of 
fungus on the upper surface of 
the specimens are reported (no 
growth, microscopic growth, 
macroscopic growth). The test 
can also be used to test the 
effect of cleaning regimes. 

Specimens (850 mm x 350 mm) 
are conditioned at 20°C and 
65% RH before being subjected 
to 2 wet and 2 dry passes using 
a commercial spray extraction 
machine or a test rig. After 24 h 
drying, 12 specimens (each 60 
mm diameter) are cut from the 
carpet. An aliquot (1 ml) of a 
suspension of cells of E. coli in 
nutrient broth is poured onto 
filter paper (7 cm diameter). 
The filter paper is then pressed 
for 1 min onto the surface of the 
carpet using a 1 kg weight. The 
filter paper is then discarded. 
After 0, 6 and 24 hours 
incubation at a specified 
temperature the carpet´s 
surface is pressed onto contact 
plates of McConkey agar. After 
24h replicate (3) plugs (10 mm ) 
are taken from each specimen 
and suspended in 10 ml nutrient 
broth for 30 seconds and then 
analysed for the presence of E. 
coli by total viable count. 

Cell suspension intimate 
contact test. 
Potential to demonstrate 
the effectiveness of an 
antimicrobial treatment if 
appropriate incubation 
conditions are selected 
and addition species 
employed. 

   
 
 
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399 

Table VIII: Methods used to Examine the Antimicrobial Activity of Non-Porous 
Surfaces 

Reference 

Title 

Description 

Major Principle 

JIS Z 2801: 
2000 

Antimicrobial 
products - Test for 
antibacterial activity 
and efficacy 

ISO 
22196:2011 

Plastics - 
Measurement of 
antibacterial activity 
on plastics surfaces. 

XP G 39-010  Propriétés des 

étoffes - Étoffes et 
surfaces 
polymériques à 
propriétés 
antibactériennes - 
Caractérisation et 
mesure de l'activité 
antibactérienne 

Cell suspension intimate 
contact test. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Cell suspension intimate 
contact test. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Cell suspension intimate 
contact test. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

The surface of replicate sample 
(3 for each treatment and 6 for 
the blank reference material - 
usually 50 mm x 50 mm) are 
inoculated with a suspension of 
either E. coli or S. aureus in a 
highly diluted nutrient broth. 
The cell suspension is then held 
in intimate contact with the 
surface by the use of a sterile 
polyethylene film (usually 40 
mm x 40 mm) for 24 hours at 
35° C under humid conditions. 
The size of the population on 
the treated surface is then 
compared with the size on the 
control surface both prior to and 
after incubation. A neutraliser 
for certain biocide types is 
employed. Antibacterial activity 
is certified if the difference 
between the Log10 of the 
population on the treated 
sample and that on the control 
surface is > 2. 

This is the current New Work 
Proposal at ISO created from 
JIS Z 2801 by the SIAA of 
Japan. Modification and 
validation is in progress in 
collaboration with the IBRG. 
Some changes are expected. 

Four replicate samples of test 
material are placed in contact 
with an agar plate that has been 
inoculated with a specified 
volume of a known cell 
suspension of either S. aureus 
and K. pneumoniae using a 
200g weight for 1 minute. The 
samples are then removed. 
Duplicate samples are analysed 
for the number of viable 
bacteria both before and after 
incubation under humid 
conditions at 37°C for 24 hours. 
A neutraliser is employed during 
cell recovery. 

ASTM 
E2180-07 

Standard Test 
Method for 
Determining the 
Activity of 

Replicate (3) samples of 
material are inoculated with 
cells of either S. aureus or K. 
pneumoniae suspended in 

Immobilised cell 
suspension intimate 
contact test. 
Basic efficacy test that 

 
 
 
 
 
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Reference 

Title 

Description 

Major Principle 

Incorporated 
Antimicrobial 
Agent(s) in Polymeric 
or Hydrophobic 
Materials 

ASTM 
E2149-10 

Standard Test 
Method for 
Determining the 
Antimicrobial Activity 
of Immobilized 
Antimicrobial Agents 
Under Dynamic 
Contact Conditions 

molten semi-solid isotonic 
saline/agar. This attempts for 
form an ‘artificial biofilm’ which 
holds the suspension in intimate 
contact with the test surface of 
inherently hydrophobic 
materials. Samples are then 
incubated at a temperature 
similar to that intended for the 
final use for a specified period 
(usually 24 hours) under humid 
conditions. The size of the viable 
bacterial populations on the 
control and treated surfaces is 
then determined using a dilution 
plate count. Any effect is 
recorded using percent 
reduction calculated from the 
geometric means of the data. A 
neutraliser may be employed 
and sonication is used to 
separate the ‘biofilm’ from the 
test surfaces and suspend the 
agar gel. Subsequent imprinting 
of the test surface onto solid 
nutrient media can be 
performed to look for the 
presence of adherent viable 
cells. 

Dynamic shake flask test. Test 
material is suspended in a 
buffer solution containing a 
known number of cells of 
Klebsiella pneumoniae and 
agitated. Efficacy is determined 
by comparing the size of the 
population both before and after 
a specified contact time. 

has limited use as a 
simulation of final use of 
a treated material. 

Relies on either diffusion 
of antimicrobial agents 
from treated material into 
the cell suspension or due 
to interaction between 
the population and the 
surface of the material in 
suspension. 
Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

   
 
 
 
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Appendix 10. Commonly  Used  Methods 
Antimicrobial Activity48 

to  Measure 

Table VI: Methods used to Examine the Antimicrobial Activity of Textiles 
(fabric, yarn or pile/wadding) 

Reference 

Title 

Description 

Major Principle 

ASTM 
E2149-10 

Standard Test 
Method for 
Determining the 
Antimicrobial Activity 
of Immobilized 
Antimicrobial Agents 
Under Dynamic 
Contact Conditions 

Dynamic shake flask test. Test 
material is suspended in a buffer 
solution containing a known 
number of cells of Klebsiella 
pneumoniae and agitated. Efficacy 
is determined by comparing the 
size of the population both before 
and after a specified contact time. 

AATCC 
147-2011 

Antibacterial Activity 
Assessment of 
Textile Materials: 

Parallel Streak 
Method 

AATCC 
100-2012 

Antibacterial Finishes 
on Textile Materials: 
Assessment of. 

Agar plates are inoculated with 5 
parallel streaks (60 mm long) of 
either Staphylococcus aureus or K. 
pneumoniae. A textile sample is 
then placed over the streaks and in 
intimate contact with the surface of 
the agar and incubated. Activity is 
assessed based on either the mean 
zone of inhibition over the 5 
streaks or the absence of growth 
behind the test specimen. 

Replicate samples (sufficient to 
absorb 1 ml of test inoculum) of 
fabric are inoculated with individual 
bacterial species (e.g. S. aureus 
and K. pneumoniae) suspended in 
a nutrient medium. The samples 
are incubated under humid 
conditions at 37° C for a specified 
contact time. Activity is assessed 
by comparing the size of the initial 
population with that present 
following incubation. A neutraliser 

Relies on either 
diffusion of 
antimicrobial from 
treated material into 
the cell suspension.  
Some activity may be 
due to interaction 
between the 
population and the 
surface of the material 
in suspension. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

Zone diffusion assay. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

Cell suspension 
intimate contact test. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

48 Please note: The methods listed are not necessarily appropriate in all cases. Their applicability 
depends  on  the  claim  made,  the  materials  used  and  the  conditions  of  use  for  the  treated 
material/article. These methods are listed to give an overview for the assessor when and where a 
method is meaningful to demonstrate a claim and where its limits are. 

 
 
 
 
 
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Reference 

Title 

Description 

Major Principle 

XP G 39-010  Propriétés des 

étoffes - Étoffes et 
surfaces 
polymériques à 
propriétés 
antibactériennes - 
Caractérisation et 
mesure de l'activité 
antibactérienne 

JIS L 1902: 
2008 

Testing Method for 
Antibacterial Activity 
of Textiles 
Qualitative Test 

JIS L 1902: 
2008 

Testing Method for 
Antibacterial Activity 
of Textiles 
Quantitative Test 

EN ISO 
20645 - 
2004 

Textile Fabrics - 
Determination of the 
antibacterial activity 
- Agar plate test 
(ISO/FDIS 
20645:2004) 

Cell suspension 
intimate contact test. 

The transfer method of 
inoculation could be 
adapted to provide 
some simulation data. 

Zone diffusion assay. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

Cell suspension 
intimate contact test. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

Zone diffusion assay. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

is employed recovery. 

Four replicate samples of test 
material are placed in contact with 
an agar plate that has been 
inoculated with a specified volume 
of a known cell suspension of either 
S. aureus and K. pneumoniae using 
a 200 g weight for 1 minute. The 
samples are then removed. 
Duplicate samples are analysed for 
the number of viable bacteria both 
before and after incubation under 
humid conditions at 37° C for 24 
hours. A neutraliser is employed 
during cell recovery. 

Three replicate samples of fabric, 
yarn or pile/wadding are placed in 
intimate contact with the surface of 
agar plates that have been 
inoculated with a cell suspension of 
either S. aureus or K. pneumoniae 
and incubated at 37° C for 24 - 48 
hours. The presence of and size of 
any zone of inhibition around the 
samples is then recorded. 

Replicate samples of fabric (6 of 
the control and 3 of the treated) 
are inoculated with individual 
bacterial species (e.g. S. aureus 
and K. pneumoniae) suspended in 
a heavily diluted nutrient medium. 
The samples are incubated under 
humid conditions at 37° C for a 
specified contact time. Activity is 
assessed by comparing the size of 
the initial population in the control 
with that present following 
incubation. No neutraliser is 
employed during cell recovery. 

Four replicate samples of fabric (25 
± 5 mm) are placed in intimate 
contact with a solid nutrient 
medium in a petri dish. The 
samples are then overlaid with 
molten solid nutrient media which 
has been inoculated with a cell 
suspension of either S. aureus, 
Escherichia coli or K. pneumoniae. 
The plates are then incubated for 
between 18 and 24 hours and the 
plates are then assessed for growth 
based on either the presence of a 
zone of inhibition of > 1 mm or the 

   
 
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403 

Reference 

Title 

Description 

Major Principle 

SN 195920 

Examination of the 
Antibacterial Effect 
of Impregnated 
Textiles by the Agar 
Diffusion Method 

SN195924 

Textile Fabrics - 
Determination of the 
Antibacterial 
Activity: 

Colony Plate Count 
Method 

SN195921 

Textile Fabrics - 
Determination of 
Antimycotic Activity: 
Agar Diffusion Plate 
Test 

Zone diffusion assay. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

Cell suspension 
intimate contact test. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

Zone diffusion assay. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

absence/strength of the growth in 
the media overlaying the test 
specimen. 

Four replicate samples of fabric (25 
± 5 mm) are placed in intimate 
contact with a solid nutrient 
medium in a petri dish. The 
samples are then overlaid with 
molten solid nutrient media which 
has been inoculated with a cell 
suspension of either S. aureus or E. 
coli. The plates are then incubated 
for between 18 and 24 hours and 
the plates are then assessed as 
described in BS EN ISO 20645 
above. 

Fifteen replicate samples (each 
replicate is comprised of sufficient 
specimens of 25 ± 5 mm to absorb 
1 ml of test inoculum) are 
inoculated with cells of either E. 
coli or S. aureus suspended in a 
liquid nutrient medium and 
incubated in sealed bottles for up 
to 24 hours at 27° C. After 0, 6 and 
24 hours, 5 replicate samples are 
analysed for the size of the viable 
population present. A neutraliser is 
employed. An increase of 2 orders 
of magnitude of the population 
exposed to a control sample is 
required to validate the test.  The 
method defines a textile as 
antibacterial if no more than a 
specified minimum level of growth 
is observed after 24 hours in 4 of 
the 5 replicate groups of samples. 

Replicate (4) samples of sterilised 
fabric (25 ± 5 mm diameter) are 
placed in intimate contact with a 
solid nutrient medium in a petri 
dish. Each petri dish has been 
prepared as a double layer. The 
first layer consists of 10 ml nutrient 
agar, the second layer of another 
10 ml of the same nutrient agar to 
which 0.1 ml spore suspension (107 
ml-1) of either Candida albicans, 
Aspergillus niger, Cladosporium 
sphaerospermum or Trichophyton 
mentagrophytes had been added. 
The plates are then incubated at 
28° C either 2 days (C. albicans) or 
7 days (A. niger, C. 
sphaerospermum and T. 

 
 
 
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Reference 

Title 

Description 

Major Principle 

ISO 20743 

Textiles - 
Determination of 
antibacterial activity 
of antibacterial 
finished products: 
Absorption method 

ISO 20743 

Textiles - 
Determination of 
antibacterial activity 
of antibacterial 
finished products: 
Transfer method 

mentagrophytes). The test is valid 
when control specimens of the 
same material without biocide, or 
of a biocide-free standard specified 
cotton material are fully 
overgrown. Good antimycotic 
efficacy is considered to be 
demonstrated when the specimens 
show no fungal growth on their 
surface. The test specifies that both 
sides of a material have to be 
tested. 

Replicate (6) samples of textile are 
inoculated with a standardised 
broth culture of either S. aureus or 
K. pneumoniae in individual tubes 
and then incubated at 37° C for 18 
- 24 hours in closed containers. 
Samples are analysed for the 
presence of viable bacteria both 
before and after incubation by 
either total viable count or the 
determination of total ATP.  
Samples are sterilised prior to 
testing and a neutraliser is 
employed during recovery.  The 
test is validated by growth of ^1 
order of magnitude during the 
incubation period. 

Replicate (6) samples of test 
material are placed in contact with 
an agar plate that has been 
inoculated with a specified volume 
of a known cell suspension of either 
S. aureus and K. pneumoniae using 
a 200 g weight for 1 minute. The 
samples are then removed. 
Replicate (3) samples are analysed 
for either the number of viable 
bacteria or the total ATM content 
both before and after incubation 
under humid conditions at 37° C 
for 24 hours. Samples are sterilised 
prior to testing and a neutraliser is 
employed during cell recovery. The 
test is validated by either growth of 
^1 order of magnitude during the 
incubation period or by a measure 
of the variability of the data 
obtained. 

Cell suspension 
intimate contact test. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

Cell suspension 
intimate contact test. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

ISO 20743 

Textiles - 
Determination of 
antibacterial activity 
of antibacterial 
finished products: 

Replicate (6) samples of test 
material are either S. aureus and 
K. pneumoniae by ‘printing’ cells 
collected on a membrane filter onto 
their surface in a standardised 

‘Dry’ inoculum 
intimate contact test. 

The transfer method of 
inoculation could be 

   
 
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Reference 

Title 

Description 

Major Principle 

Printing method 

ISO/FDIS 
13629-1 

Textiles - 
Determination of 
Antifungal Activity of 
Textile Products: 
Part 1 - 
Luminescence 
Method 

ISO/WD 
13629-1 

Textiles - 
Determination of 
Antifungal Activity of 
Textile Products: 
Part 2 - Plate Count 
Method 

adapted to provide 
some simulation data. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

The transfer method of 
inoculation could be 
adapted to provide 
some simulation data. 

Basic efficacy test that 
has limited use as a 
simulation of final use 
of a treated material. 

The transfer method of 
inoculation could be 
adapted to provide 
some simulation data. 

manner. The samples are then 
incubated under humid conditions 
for 18 - 24 hours at 20° C for a 
specified contact time(s). Replicate 
(3) samples are analysed for either 
the number of viable bacteria or 
the total ATM content both before 
and after incubation. Samples are 
sterilised prior to testing and a 
neutraliser is employed during cell 
recovery. The test is validated by 
either determining the survival of 
the inoculum on the control 
material. 

Samples of textiles are inoculated 
with a suspension of fungal spores 
either by direct application or 
transfer from an agar surface and 
then incubated. Germination and 
growth of the spores is followed by 
measuring the ATP concentration 
associated with the samples. The 
presence of an antifungal 
treatment is expected to show 
either an inhibition of germination 
or a reduction in the rate of growth 
as indicated by reduced 
concentrations of ATP associated 
with the treated material in 
comparison with the untreated 
material. 

Samples of textiles are inoculated 
with a suspension of fungal spores 
either by direct application or 
transfer from an agar surface and 
then incubated. Germination and 
growth of the spores is followed by 
measuring the number of colony 
forming units. The presence of an 
antifungal treatment is expected to 
show either an inhibition of 
germination or a reduction in the 
rate of growth as indicated by 
reduced numbers of colony forming 
units associated with the treated 
material in comparison with the 
untreated material. 

 
 
 
 
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Table VII: Methods used to Examine the Antimicrobial Activity of Carpets 

Reference 

Title 

Description 

Major Principle 

AATCC 
174-2011 

Antimicrobial 
Activity 
Assessment of 
Carpets 

Qualitative 
Antibacterial 
Activity 

AATCC 
174-2011 

Antimicrobial 
Activity 
Assessment of 
Carpets 

Quantitative 
Antibacterial 
Activity 

AATCC 
174-2011 

Antimicrobial 
Activity 
Assessment of 
Carpets 

Quantitative 
Antifungal Activity 

Petri dishes with nutrient media are 
inoculated with a single, diagonal 
streak (approx.7.5 cm) of either S. 
aureus or K. pneumoniae. An 
unsterilized test specimen (25 mm 
x 50 mm) is placed in intimate 
contact and transversely across the 
inoculum on the agar surface. The 
plates are then inoculated at 37° C 
for 18 - 24 hours. The front and 
back of the carpet are tested 
separately. After incubation, the 
plates are inspected for the 
presence of growth both below the 
specimens and for any zone of 
inhibition surrounding the 
specimens. The test can also be 
used to test the effect of cleaning 
regimes. An untreated control is 
optional. 

Unsterilized specimens of carpet 
are pre-wetted with either sterile 
water or a wetting agent before 
being inoculated with individual 
suspensions of either S. aureus or 
K. pneumoniae in either a low or a 
high nutrient solution. The samples 
are then incubated in a tightly 
closed jar at 37° C for a specified 
contact time. Cells are recovered in 
100 ml of a neutraliser after 0 and 
6 - 24 hours of incubation. Activity 
is assessed by comparing the size 
of the initial population in the 
control (if used) with that present 
following incubation. A control is 
optional. When not employed, 
viable counts following incubation 
of the treated specimens alone are 
considered.  The test can also be 
used to test the effect of cleaning 
regimes. 

Petri dishes containing Sabouraud 
Dextrose Agar are inoculated with 
1 ml of a spore suspension of 
Aspergillus niger. Immediately 
afterwards, specimens (38 mm 
diameter) of unsterile test material 
are placed into intimate contact 
with the agar. An additional 0.2 ml 
of the same spore suspension is 
also employed to inoculate the test 

Qualitative assessment of 
rate of kill and zone 
diffusion test 

Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Cell suspension intimate 
contact test. 

Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Zone diffusion 
test/surface growth test. 

Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

   
 
 
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Reference 

Title 

Description 

Major Principle 

WIRA Test F  Test Method for 

Assessing the 
Survival of Test 
Organisms on 
Floor Coverings 

pieces directly. The samples are 
then incubated at 28°C for 7 days. 
The back and front of the discs of 
carpet are tested in separate 
dishes. The zone of inhibition and 
the growth of fungus on the upper 
surface of the specimens are 
reported (no growth, microscopic 
growth, macroscopic growth). The 
test can also be used to test the 
effect of cleaning regimes. 

Specimens (850 mm x 350 mm) 
are conditioned at 20°C and 65% 
RH before being subjected to 2 wet 
and 2 dry passes using a 
commercial spray extraction 
machine or a test rig. After 24 h 
drying, 12 specimens (each 60 mm 
diameter) are cut from the carpet. 
An aliquot (1 ml) of a suspension of 
cells of E. coli in nutrient broth is 
poured onto filter paper (7 cm 
diameter). The filter paper is then 
pressed for 1 min onto the surface 
of the carpet using a 1 kg weight. 
The filter paper is then discarded. 
After 0, 6 and 24 hours incubation 
at a specified temperature the 
carpet´s surface is pressed onto 
contact plates of McConkey agar. 
After 24h replicate (3) plugs (10 
mm) are taken from each specimen 
and suspended in 10 ml nutrient 
broth for 30 seconds and then 
analysed for the presence of E. coli 
by total viable count. 

Cell suspension intimate 
contact test. 

Potential to demonstrate 
the effectiveness of an 
antimicrobial treatment if 
appropriate incubation 
conditions are selected 
and addition species 
employed. 

 
 
 
 
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Table VIII: Methods used to Examine the Antimicrobial Activity of Non-Porous 
Surfaces 

Reference 

Title 

Description 

Major Principle 

JIS Z 2801: 
2000 

Antimicrobial 
products - Test 
for antibacterial 
activity and 
efficacy 

Cell suspension intimate 
contact test. 

Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

The surface of replicate sample (3 
for each treatment and 6 for the 
blank reference material - usually 
50 mm x 50 mm) are inoculated 
with a suspension of either E. coli 
or S. aureus in a highly diluted 
nutrient broth. The cell suspension 
is then held in intimate contact 
with the surface by the use of a 
sterile polyethylene film (usually 40 
mm x 40 mm) for 24 hours at 35° 
C under humid conditions. The size 
of the population on the treated 
surface is then compared with the 
size on the control surface both 
prior to and after incubation. A 
neutraliser for certain biocide types 
is employed. Antibacterial activity 
is certified if the difference between 
the Log10 of the population on the 
treated sample and that on the 
control surface is > 2. 

ISO 
22196:2011 

Plastics - 
Measurement of 
antibacterial 
activity on 
plastics surfaces. 

This is the current New Work 
Proposal at ISO created from JIS Z 
2801 by the SIAA of Japan. 
Modification and validation is in 
progress in collaboration with the 
IBRG. Some changes are expected. 

Cell suspension intimate 
contact test. 

Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

XP G 39-010  Propriétés des 

étoffes - Étoffes 
et surfaces 
polymériques à 
propriétés 
antibactériennes - 
Caractérisation et 
mesure de 
l'activité 
antibactérienne 

Four replicate samples of test 
material are placed in contact with 
an agar plate that has been 
inoculated with a specified volume 
of a known cell suspension of either 
S. aureus and K. pneumoniae using 
a 200g weight for 1 minute. The 
samples are then removed. 
Duplicate samples are analysed for 
the number of viable bacteria both 
before and after incubation under 
humid conditions at 37°C for 24 
hours. A neutraliser is employed 
during cell recovery. 

Cell suspension intimate 
contact test. 

Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

ASTM 
E2180-07 

Standard Test 
Method for 
Determining the 
Activity of 
Incorporated 

Replicate (3) samples of material 
are inoculated with cells of either 
S. aureus or K. pneumoniae 
suspended in molten semi-solid 
isotonic saline/agar. This attempts 

Immobilised cell 
suspension intimate 
contact test. 

   
 
 
 
 
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Reference 

Title 

Description 

Major Principle 

Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

Antimicrobial 
Agent(s) in 
Polymeric or 
Hydrophobic 
Materials 

for form an ‘artificial biofilm’ which 
holds the suspension in intimate 
contact with the test surface of 
inherently hydrophobic materials. 
Samples are then incubated at a 
temperature similar to that 
intended for the final use for a 
specified period (usually 24 hours) 
under humid conditions. The size of 
the viable bacterial populations on 
the control and treated surfaces is 
then determined using a dilution 
plate count. Any effect is recorded 
using percent reduction calculated 
from the geometric means of the 
data. A neutraliser may be 
employed and sonication is used to 
separate the ‘biofilm’ from the test 
surfaces  and suspend the agar gel. 
Subsequent imprinting of the test 
surface onto solid nutrient media 
can be performed to look for the 
presence of adherent viable cells. 

ASTM 
E2149-10 

Standard Test 
Method for 
Determining the 
Antimicrobial 
Activity of 
Immobilized 
Antimicrobial 
Agents Under 
Dynamic Contact 
Conditions 

Dynamic shake flask test. Test 
material is suspended in a buffer 
solution containing a known 
number of cells of Klebsiella 
pneumoniae and agitated. Efficacy 
is determined by comparing the 
size of the population both before 
and after a specified contact time. 

Relies on either diffusion 
of antimicrobial agents 
from treated material into 
the cell suspension or due 
to interaction between 
the population and the 
surface of the material in 
suspension. 

Basic efficacy test that 
has limited use as a 
simulation of final use of 
a treated material. 

 
 
 
 
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Appendix 11. Information  on 
organisms for PT 8 as outlined in the document (5.5.8) 

the  principle 

target 

Fungi  

Wood rotting fungi 

White rot/ brown rot fungi (Basidiomycetes): 

Fungi responsible for brown rot (e.g. Serpula lacrymans, Coniophora puteana) and white 
rot (e.g. Coriolus versicolor, Donkioporia expansa) 

Soft rot fungi (mainly Ascomycetes, Deuteromycetes): 

Fungi  responsible  for  a  type  of  rot  characterised  by  surface  softening  of  the  wood 
although they also cause rot at depth (e.g. Chaetomium globosum). They are specifically 
significant for wood in ground contact. 

Wood discolouring fungi 

Sapstain: 

The  blue-black  and  brown  discolouration  of  freshly  felled  logs  or  sawn  timber  have  an 
economic importance. Sapstain causing fungi can only colonise wood as long as the sap 
wood  contains  enough  water  to  provide  solved  sugars  as  a  nutrient  for  these  fungi 
("green"  wood).  Therefore,  these  fungi  can  be  controlled  by  rapid  drying  of  the  wood 
after felling, chemical treatments are sometimes used. 

Common sapstain species include e.g. Stereum spp., blue staining species. 

Blue stain cause blue to black permanent colour of variable intensity and depth mainly in 
the  sapwood  depending  on  the  wood  species.  This  does  not  result  in  appreciable 
alteration  of  the  mechanical  properties  but  can  increase  the  permeability  of  the  wood 
and thereby makes it more susceptible to fungal degradation.  

Common blue staining species include e.g. Aureobasidium spp., Ceratocystis spp. 

Mould fungi: 

Fungi, e.g. Aspergilus spp., Penicillium spp. being evident as spots of various colours on 
the  surface  of  moist  wood.  (for  instance,  as  a  result  of  high  relative  humidity  or  of 
condensation of water vapour). They do not significantly alter the mechanical properties 
of  the  wood  but  have  a  special  significance  for  wood  in  service  if  discoloration  is 
undesirable or unacceptable. 

For green sawn timber, the moulds are covered by the CEN TS 15082 standard. But for 
the preservation of solid wood against mould, the EN 152 does not cover mould and no 
CEN  standard  is  available.  In  that  case  the  applicant  is  invited  to  submit  relevant  data 
(in house method, literature data...) which could be accepted by expert judgement. 

Insects  

Fresh wood insects 

A  number  of  insects  bore  and  tunnel  into  fresh  logs  after  they  are  cut  and  debarked. 
These fresh wood insects feed upon the  starch reserves and  can cause damages to the 
wood. Most of them belong to the families of Scolytidae (genus Scolytus), Cerambycidae 
(genus Phematodes), Lyctidae (genus Lyctus), Anobiidae (genus Anobium), Bostrychidae 
(genus Bostrychus). 

Some other groups, belonging to the Scolytidae family, bore the fresh logs and introduce 
‘Ambrosia’  fungi  inside  the  gallery,  resulting  in  wood  staining  (as  a  consequence  of  the 
development of the dark hyphae). 

   
 
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Wood boring beetles (Coleoptera)  

Insects which lay their eggs in wood pores or cracks and whose larvae feed upon wood. 
They  are  present  throughout  Europe  but  the  risk  of  attack  varies  greatly  and  is  ranged 
from  high  to  insignificant.  The  most  important  are  Hylotrupes  bajulus,  Anobium 
punctatum and Lyctus brunneus. 

Hylotrupes bajulus (House longhorn beetle) 

This beetle attacks many softwood species and can cause significant structural damage. 
Many  softwood  species  are  affected,  whereas  hardwoods  are  not  attacked.  Larvae 
damage both the sapwood and the heartwood of non durable species. 

This insect  occurs throughout Europe, but is  of less importance in the north and north-
west  of  Europe.  The  vitality  and  longevity  of  larvae  depend  principally  on  ambient 
temperature and the wood moisture content. 

Anobium punctatum (Common furniture beetle) 

The  larvae  attack  the  sapwood  of  certain  softwood  and  hardwood  species.  The  damage 
can  extend  to  the  heartwood  in  some  wood  species  and  can  have  occasionally  a 
structural  significance  impact.  Its  presence  is  particularly  noted  in  coastal  climates  and 
where damp conditions prevail. 

Lyctus brunneus (Powder post beetle) 

The larvae attack sapwood of certain starch-containing hardwoods and have a significant 
impact throughout Europe for both European and imported hardwood timbers. 

Termites (Isoptera) 

Termites belong to the order Isoptera. In Europe and in the European tropical overseas 
regions there are three main termite families; subterranean termites (Rhinotermitidae), 
drywood termites (Kalotermitidae) and tree termites (Nasutitermitidae): 

- 

Reticulitermes is the most common genus encountered from the Rhinotermitidae 
family  in  Europe.  The  main  species  registered  are:  R.  flavipes  (former  R.  santonensis), 
R. grassei, R. lucifugus, R. banyulensis, R. balkanensis, R. urbis. 

They  are  widespread  around  the  Mediterranean  basin  (Spain,  France,  Italy,  Portugal, 
Balkans,  and  Greece)  and  Black  Sea  (Turkey,  Romania),  though  some  termite  spots  in 
the  UK  or  Germany  have  been  reported.   Several  unanswered  questions  remain  about 
the  origin  of  these  termites.   While  some  Reticulitermes  are  native  to  Europe,  others 
may be related to species from eastern North America and the Middle East (Israel, Asian 
Turkey, etc.). 

Coptotermes  and  Heterotermes  are  the  main  two  genera  belonging  also  to  the 
Rhinotermitidae family located in the European tropical overseas regions. 

Kalotermes  flavicollis  and  Cryptotermes  brevis  are  the  main  two  species  of 
drywood  termites  present  in  Europe  (especially  in  the  coastal  areas  of  Mediterranean 
countries  and  Canary  Islands).  Cryptotermes  is  a  main  genus  belonging  to  drywood 
termites encountered in the European tropical overseas regions. 

Nasutitermes is the main genus belonging to the Termitidae family (tree termites) 

encountered in the European tropical overseas regions. 

- 

- 

 
 
 
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Marine borers  

This  term  is  applied  to  marine  invertebrates  such  as  Limnoria  spp.  and  Teredo  spp. 
which  need  a  certain  salinity  of  water  and  which  hollow  out  extensive  tunnels  and 
cavities  in  wood.  These  organisms  can  cause  serious  damage  to  fixed  or  floating 
structures. 

In European waters the most common marine borers are shipworm (Teredo navalis) and 
gribble  (Limnoria  spp.).  Shipworm  is  a  bivalve  mollusc  related  to  the  sea  snails  and 
mussels. It is a soft, worm like animal with its shell modified into hard grinding jaws. The 
larvae  are  part  of  the  microscopic  zooplankton  and  swim  freely  in  the  sea  until  they 
settle  on  timber.  They  develop  a  shell  with  which  they  bore  into  the  wood  and  lodge 
there, growing into large worms in holes up to 5 mm in diameter. They destroy the wood 
by  making  a  massive  network  of  galleries  throughout  the  timber.  Gribble  is  a  small 
shrimp-like crustacean about 4 mm in length. It bores into the surface of the wood and 
lodges near the surface making numerous side burrows. The combination of this boring 
and wave action causes rapid erosion of marine timbers. 

   
 
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Appendix 12. Annex A of EN 599-1 

Introduction 

Additional explanations regarding Annex A of the standard EN599-1:2009+A1 :2013, 
mainly on cases where no new biological testing is necessary, are presented here 
following the sections of the Annex. 

NOTES to the reader:  

Annex A is not a normative Annex in EN 599-1 but only informative and intended to 
act as guidance. 

Annex A of EN 14128: the majority of the points (below) also apply to Annex A of EN 
14128,  except  for  Section  A.3;  additional  notes  will  be  added  at  a  future  update  of 
this guidance document.  

In  this  Appendix,  a  “ready-to-use  formulation”  refers  to  the  product  as  marketed; 
this  includes  concentrated  products  (which  are  diluted  before  application)  and 
products  which  do  not  need  to  be  diluted  before  application,  (i.e.  they  can  be  used 
directly  from  the  container).  The  efficacy  will  be  demonstrated  at  the  concentration 
used. 

The introduction to Annex A lists the modifications which can occur during the 
development of a product for the first or subsequent authorisations (e.g. minor change, 
major change). 

The composition of the products as tested is the basis to assess the variations that can 
occur in a biocidal product family (BPF). 

Variations can occur within a BPF which fall outside some of the guidelines given in 
Annex A and which may require additional efficacy testing even if the products are 
considered within the same BPF; nor should Annex A be considered as being only 
applicable to a BPF. 

This Appendix has been written to give guidance on whether existing test results could 
still be considered valid where formulation changes have been made or when additional 
laboratory testing according to the provisions of EN 599 may be required. This is a 
helpful and pragmatic approach regardless of whether a BPF is being considered or a first 
or subsequent authorisation. 

Sections of Annex A– additional explanation 

Paragraph A.2 No requirements for new biological testing 

Section A.2.1 

This section lists all the allowed variations from the tested formulation of the BP (given 
in sub-sections A.2.2, A.2.3 and A.2.4), for which no new biological testing is required. It 
should be clarified if only one variation is allowed between the two products or if several 
variations are allowed. 

→   all of the variations may occur (and are allowed). 

Section A.2.2 In the case of organic solvent based products (ready for use) 

o  Sub-section A.2.2 a 

Changes involving substitution of any co-formulant by one which is 
chemically equivalent from another supplier. 

 
 
 
 
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→   “Substitution” means replacement of a chemically equivalent co-formulant 
performing the same function in the product formulation. 

“Chemically equivalent” means that chemicals have the same CAS number and the 
same physical properties (e.g. pH, molecular weight distribution (for polymers), 
HNL number (for surfactants). It is a chemical from another supplier. 

Information on function of the co-formulant should be provided; co-formulants are 
any ingredient other than an active ingredient, in a formulated wood preservative 
product. Typical chemical functions for non-active ingredients of wood 
preservative can be, for example, solvents, surfactant, emulsifier, corrosion 
inhibitor, binder, pH stabiliser, mordant, dye, pigment, ‘penetration marker’ water 
repellent and co-solvent. 

o  Sub-section A.2.2 b 

Products to be applied by penetrating treatment processes for changes in 
the aromatic content or chemical nature of hydrocarbon solvent 
carriers49, providing that not less than 90% (v/v) of the carrier distils 
below  250°C. 

→   The reason for such a change is that a product could have been tested (e.g. 
in an EN113 test) using the organic solvent xylene. The xylene evaporates during 
drying of the treated blocks leaving the active substance in the dry wood blocks, 
so the solvent does not affect the efficacy of the product. The blocks are exposed 
to the test fungus and the Biological Reference Value (BRV) and the Critical Value 
(CV) for the product are determined. This point in Annex A allows an organic 
solvent based product containing the active substance to be formulated (using an 
aromatic substance such as ‘Caromax 18’, ‘white spirit’, ‘Stoddard solvent’ or 
‘odourless kerosene’ to dissolve the active substance) without retesting the 
product. The principle is that the organic solvent evaporates after treatment, 
leaving the product solid at, or above, the CV and the type and composition of the 
organic solvent carrier does not affect the efficacy of the product. Thus an efficacy 
test of a product (e.g. EN 113, EN 47) with a xylene solvent/carrier can be used 
to confirm the efficacy of an organic solvent based product applied by a 
penetrating process with a different solvent carrier, providing that not less than 
90% (v/v) of the carrier distils below 250oC. If the data are not available (e.g. the 
SDS of an hydrocarbon solvent carrier does not contain information about boiling 
point), a justification should be provided by the applicant. 

o  Sub-section A.2.2 c 

Product to be applied by superficial processes, for a change in the 
aromatic content of hydrocarbon solvent carriers of no greater than 10% 
(v/v of the total aromatic hydrocarbon solvent content). 

→   See point A.2.2 point b. 

Example: 

A formulation tested (e.g. EN113) with 20% m/m aromatic hydrocarbon 
solvent/carrier, can be read across to a biocidal product containing no less than 

49 The carrier is the substance used to convey the wood preservative formulation into the wood 
e.g. water or organic solvent (see for example EN 599-1 A2.2(b)). A solvent may be used as the 
main carrier but may also be used for other purposes within the wood preservative formulation 
(e.g. as part of a micro-emulsion).  In the latter case, EN 599-1 refers to this as a “co-solvent” 
(see for example EN 599-1 A2.3(f)). 

   
 
 
 
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18% and no more than 22% total aromatic hydrocarbon solvent (= ± 10% of 
20%). 

o  Sub-section A.2.2 d + e 

•  Changes involving the addition or deletion of a soluble dyestuff.  

•  Changes in pigments to an equal or lower pigment content of the 

product.  

→  “Soluble dyestuffs” (‘dyes’ in the BPR) are coloured, non-biocidal soluble 
substances which do not impede the flow of liquid through the wood structure; 
this is so that they do not reduce penetration of the active substances in a wood 
preservative and do not affect the efficacy of an active substance or biocidal 
product. Dyes may be included in a wood preservative as a penetration marker to 
differentiate between treated and untreated timber and/or to colour the preserved 
wood. 

 “Pigments” are coloured, non-biocidal, insoluble materials, dispersed in a 
suitable medium. Some pigments have been found to reduce the penetration of 
the active substances in a wood preservative. 

Due to the potential impact of pigments on penetration it was decided to allow 
changes only up to the former content of pigment (solid portion) in the 
formulation when the ‘no additional testing rule’ must apply. If the exact content 
of the pigment and its solid portion is unknown changes up to the total content of 
the pigment paste are allowed if robust justification is provided. 

It can be accepted to test a formulation without pigment. 

In cases where additional pigments are used in the product, it has to be 
demonstrated that the conditions of A.2.5 are fulfilled. 

o  Sub-Section A.2.2 f 

Product containing 10% (m/m) or less of solids containing resins and/or 
water repellents50, relative changes in content of these constituent(s) of 
no more than ± 20% (m/m) and products containing more than 10% 
(m/m) solids, relative changes of no more than ± 10% (m/m).  

→   With reference to wood preservative formulations, a solid is the proportion of 
non-volatile material contained in a formulation after the volatile solvent, (which 
serves as a carrier or vehicle for the solid content) has vaporized or evaporated. 

A “resin” is a non-volatile organic polymer and can be solid, semi-solid or liquid 
form. 

An ingredient can be considered to make up the ‘solid’ portion of the preservative 
if it is non-volatile. However, in this section the solid content being referred to are 
specifically resins plus water repellents. Solids of pigments are excluded here and 
dealt with in A.2.2e. 

Example of a calculation of the allowed variations in case of a product containing 
resin and water repellent: 

For a product containing 5% resin + 7% water repellent (non-volatile portion) 
then the allowed variation is: (5+7) * 10 / 100 = ± 1.2%. 

50 Water repellents are co-formulants in a formulation impart additional resistance to the 
absorption of water by the treated wood product. Typically water repellents are, but not limited to, 
of waxes or silicon base. 

 
 
 
 
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o  Sub-Section A.2.2 g 

Up to 5% of the hydrocarbon solvent may be replaced by a solvent 
miscible co-solvent fulfilling the distillation range given in sub-Section 
A.2.2 b 

If the content of pigments in a formulation is not available (e.g. not provided in 
the SDS), the content of the pigment mixture should be applied. For products 
that cannot benefit from this approach, additional data on the solid pigment 
content should be requested from the applicant. 

o  Sub-Section A.2.2 h 

Adding and/or replacing a co-formulant providing the additive 
constitutes less than 2% of the total formulation and providing the 
physical properties are not affected (A.2.5). 

→“Replacing” means changing one co-formulant for another. Partial replacement 
is permissible. 

”Adding” refers to both the addition of a new co-formulant and to the increase of 
an existing co-formulant. 

The 2% relates to each individual substance. This value was chosen on the basis 
that it represents a safe level of change within a formulation that experts were 
confident would not affect the efficacy of a formulation, provided that stability 
was unaffected (hence the requirement that the provisions in A.2.5 should be 
met). 

An example of formulation modification to illustrate this section could be the 
exchanged/amended amount of propylene glycol with ethylene glycol by a change 
of ± 2%. 

Section A.2.3 In the case of water-soluble preservatives 

o  Sub-section A.2.3 a: see A.2.2 a 

o  Sub-section A.2.3 b: see A.2.2 d 

o  Sub-section A.2.3 c 

For products in their ready for use form containing 10% (m/m) or less of 
solids containing resins and/or water repellents, relative changes in 
content of these constituent(s) of no more than ± 20% (m/m) and for 
products containing more than 10% (m/m) solids, relative changes of no 
more than ± 10% (m/m)of these constituents. 

See also Sub-section A.2.2 f for the definitions of “solid” and “resin”. 

o  Sub-section A.2.3 d 

In a case of inorganic active substances (e.g. copper II salts), no additional 
biological testing is required when changing the inactive component (the anion 
part) of the active substance not resulting in a change in the ratio, total content 
or chemical properties of biocidal active component (e.g. copper II). 

o  Sub-section A.2.3 e: see A.2.2 e 

o  Sub-section A.2.3 f 

Changing or adding a water miscible co-solvent (distillation ranged as in 
A.2.2 b) up to 5% of the total formulation. 

o  Sub-section A.2.3 g: see A.2.2 h 

   
 
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Section A.2.4 In the case of emulsion products 

→  Differentiation between water soluble preservative (2.3) and emulsion 
products (2.4) Often products are part suspension and part emulsion. 

At the time of the development of EN 599, emulsion concentrates were a 
relatively new technology. This explains why all the comparisons were made in 
relation to water-borne preservatives. With the knowledge and widespread 
experiences nowadays this separation is not justified anymore. It is 
recommended that section A2.3 is used for all water-based preservative 
formulation types (i.e. solution/emulsion/suspension or combinations of these) 
while ensuring the physical form of the active substance in the formulation is 
unchanged (i.e. solution/emulsion/suspension). 

See section A.2.3. 

Section A.2.5. 

o  For the sub-sections A.2.2 h, A.2.3 g, , it should be confirmed that: 

• 

• 

the penetration into the wood is not adversely affected (only for 
penetrative treatments); 

the stability of the product is not adversely affected; this can be 
demonstrated e.g. with the chemical analysis, of the active 
ingredients after storage stability.  

→   If, after a formulation change, an improvement in stability is recorded 
through chemical analysis after storage at 40 degrees C, this in itself will not 
result in a requirement for additional testing. 

→   You cannot generally predict the penetration of a wood preservative product 
from its composition. The combination of product composition and application 
process governs the wood preservative penetration. 

Laboratory scale or pilot plant trials using standard timber species and standard 
process cycles would be appropriate to demonstrate that the penetration into the 
wood is not adversely affected.  

Accelerated storage stability tests can be used to fulfil the requirements for the 
stability of the product and active substance content after storage. 

Paragraph A.3 Requirement for minimum new biological testing 

Practical case: Is it possible to combine section A.3 and A.2? 

Example: 

•  Product A is a fungicidal and insecticidal product. Data on the efficacy of 

this product is available; 

•  Product B is insecticidal only and the composition is very close to the 
product A except the fungicide active substance deleted and one 
compound added to the formulation A (at 1.5% w/w). 

When it is taken into account that efficacy data demonstrate that the 
fungicidal active substance has no impact on the insecticidal active 
substance and that the point A.2.3 and A.2.5 are fulfilled. Is the double 
read across acceptable? 

•  A2 and A3 are for different situations; 

 
 
 
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•  A2 specifies conditions where there is no requirement for new biological 

testing; 

•  A3 specifies conditions for minimum new biological testing (though in the case 
of changes to fungicide and insecticide levels it also describes instances where 
no additional testing will be required). 

In the example, the data provide sufficient demonstration of the effectiveness of Product 
B against insects. 

The ‘double read across’ is acceptable. The results from the insect efficacy studies for 
Product A can be read across to Product B according to Annex A of EN599. This is 
acceptable because the addition of the compound to Product A is less than 2% w/w. 
Assuming that the description of the function of the compound in the ‘Identity’ section is 
acceptable under Annex A, because it will not adversely affect penetration, Product B 
does not require retesting under Annex A and Product B can be considered to be 
effective against insects under BPR. 

Under Annex A, the fungicidal active substances could be omitted from Product B without 
retesting the efficacy of Product B against insects if data exist which confirm that the 
removal of the fungicide does not affect the insecticidal efficacy (section A.3.2.2). 
Product B (without fungicide) can only be claimed to be effective against insects, and the 
insect studies for Product A can be used to confirm the effectiveness of Product B against 
insects. 

   
 
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Appendix 13. Laboratory  studies  for  rodenticides  :  bait 
choice test 

This appendix describes a protocol of a laboratory study to determine the efficacy of an 
as yet unauthorised product (rodenticide) against the house mouse, brown rat and roof 
rat containing a bait formulation. This protocol can be applied to other target organisms 
(e.g. voles). 

A feeding test is conducted to determine the extent to which rodents will eat the product 
when they are given a free choice between that and their normal food. This type of 
palatability test is most suited to slow-acting toxicants. The test consists of an 
acclimatisation period, followed by a pre-test diet take assessment, then a test period of 
normally51 3-5 days and at least 14 days of post-treatment observation. 

Pre-test period 

For the test, normally 10 wild or laboratory strain rodents (5 males and 5 females) are 
required. Laboratory rodents should be healthy, non-pregnant adults of known strain 
(STATE). Preferably wild adult rodents are used. They should be healthy and obtained 
from free-living populations (STATE WHERE) in accordance with Directive 2010/63/EU, 
Articles 7 and 9 and Section A, 3.2 of Annex III . On arrival at the laboratory, the wild 
strains should be treated with an appropriate insecticide to kill ectoparasites and then be 
housed in small groups (no more than five per cage) of the same sex and treatment 
group if no aggressive behaviour is expected, preferably in solid floor cages with 
appropriate environmental enrichment. Animals may be housed individually only if 
scientifically justified. With wild rats especially, it is advisable to place all items (i.e. food 
pots) required for the test in the cage before each animal is released into it. Wild rodents 
should be acclimatised to laboratory conditions for at least 3 weeks to ensure that no 
females are pregnant when the test begins. During this time they should be offered a 
laboratory animal diet and water should be freely available. To encourage variation in 
response, animals with body weights throughout the range normally expected for the 
species should be used as far as possible. 

Before the test period begins, it is necessary to ensure that the animals are feeding 
normally. Following acclimatisation, two food pots, placed either side at the front of the 
cage, are filled with cereals, such as wheat, broken wheat, or a wheat-based mixture or 
ground laboratory diet or EPA meal. All other food is removed, but water remains freely 
available. The quantity of food placed in each pot (STATE) should be sufficient to meet 
each animal’s daily needs. Food uptake should be determined, therefore all unused food 
(i.e. food left in the pot) and scattered food must be collected and taken into account by 
weighing to determine how much of the food has not been eaten. All unused diet (i.e. 
food left in the pot and scattered food) should be discarded and the pot refilled with a 
fresh supply, to ensure it is palatable. This procedure should be repeated for a further 3 
days and on the last day (of this pre-treatment period) the animals should be weighed. 
Also on the last day, the diet remaining in each pot and scattered food, is weighed and 
the total amount of food eaten by each rodent calculated (STATE). Any rodent not eating 
normally by the last day should be discarded. 

Test period 

The palatability test commences with 2 clean bait containers, one filled with a quantity of 
the test product and the other with a suitable challenge diet (e.g. an EPPO challenge 

51 Deviation from this norm is possible but should be explained in the application. 

 
 
 
 
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diet52  or standard laboratory diet). Again, the quantity in each pot should exceed the 
normal daily requirement for each animal. After 24 hours, the diet remaining in each pot 
is weighed and the total amount of food eaten by each rodent calculated. All used test 
and challenge diet is discarded and fresh quantities of each diet are placed in clean pots. 
In placing the pots back in the cage, the positions of the rodenticide and the challenge 
diet should be interchanged to avoid place preference. This procedure should be 
repeated every day during the choice period. After day 4 (3 or 5 is also acceptable) the 
animals should be returned to the standard laboratory diet. 

Observation period 

During the observation period the rodents are observed at least once per day and any 
signs of toxicity and mortality are recorded. Humane end-points should be applied in line 
with Directive 2010/63/EU to all animals showing clinical signs that can determine 
impending death. 

Guidance Document on the recognition, assessment and use of clinical signs as humane 
endpoints for experimental animals used in safety evaluation (OECD, 2002) must be 
considered. 

Results 

Results should be shown as the percentage intake of rodenticide and the percentage 
intake of challenge diet (see section 2.2.1 for further details). Also the percentage 
mortality and any other symptoms should be mentioned. 

Liquid bait formulations 

The test must be carried out as above with the following exceptions: 

•  a suitable compounded laboratory diet shall be freely available; 

• 

tap water must be used as the control bait; 

•  all procedures relating to the solid control and test baits must be applied instead 

and as appropriate to the liquid control and test baits; 

•  when the positions of the test and control baits are interchanged the positions of 

the drinking tubes, if used, should not be interchanged; 

• 

liquid baits must be provided in containers with non-drip nozzles or suitable open 
pots; 

•  a filled container must be placed out of reach of the animals in order to monitor 

weight loss due to evaporation. 

52  EPPO  guideline  PP1/113  for  the  efficacy  of  rodenticides,  Laboratory  tests  for  evaluation  of  the 
toxicity and acceptability of rodenticides and rodenticides preparations. Revised 1998. 

   
 
 
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Appendix 14. Field trial for rodenticide baits 

This appendix describes a protocol and factors to be taken into account when conducting 
a field trial to determine the efficacy of an as yet unauthorised rodenticide bait product 
against the house mouse, brown rat or roof rat. This protocol can be applied to other 
target organisms (e.g. voles). 

Ideally field trials should: 

•  be conducted with separate rat and mice populations (as appropriate to the 

intended uses in the draft SPC); 

•  be carried out at sites that are representative of the intended uses in the draft 

SPC (for example industrial, commercial, domestic); 

• 

include sites with ‘known’ anticoagulant resistant populations (if appropriate to 
the intended uses in the draft SPC); 

•  have had no rodenticide treatments over the past 6 weeks; 

• 

• 

• 

Incorporate lag phases before and after the treatment phase; 

for testing concentrates, cover a range of bait bases; 

for product that is sold with a specific bait station, include the whole device (the 
bait and its station) in the test; 

•  be carried out at 2 or 3 locations (i.e. a trial site sufficiently far away from the 

next, dependent on the roaming pattern of the test organism; e.g. Sites >30 m 
apart for Norway rats (Buckle and Smith 2015). 

The following suggested method for bait formulations details the extent of the data 
required, but the methods may be replaced or supplemented by new techniques as 
appropriate. 

Suggested procedure for bait formulations 

Trial sites 

Each trial site should, as far as possible, comprise a discrete infestation of one target 
species, with little chance of rapid reinvasion from adjoining areas. 

During the entire trial, the baiting sites should be at exactly the same locations, taking 
into account distances as specified in the intended use, local structure and rodent 
activity as established prior to the trial. See also the Good Practice Document released 
by Cefic (http://www.cefic.org/Documents/Industry%20sectors/EBPF/ 
Guideline-on-Best-Practice-in-the-Use-of-Rodenticides-in-the-EU.pdf), and the field trial 
protocol released by the RRAC (www.rrac.info/releases/technical-monographs/). 

At each baiting site, a bait container is placed, the top of which is closed/covered, to 
protect the bait from weather and avoid spillage. When selecting baiting sites, it is 
important that the animals can feed without being disturbed. 

The amount of bait applied in each feeding point should correspond to the amount given 
in  the use instructions in the draft SPC. In general, for mice, the amount of bait applied 
in each feeding point is less than for brown or roof rats. In other respects, the test 
design is identical for both groups. It is important that there is always enough fresh food 
or bait containing the active substance present. 

Before the trial begins, draw a sketch map showing all significant features of the site 
including signs of infestation. 

Data on field efficacy is likely to be more reliable if infestations of brown rats and  
house mice are selected on the basis that a stable level of activity is obtained during the 
pre-treatment assessment. The level of activity can be determined by two of the 
following (as appropriate to the situation, species etc.): 

 
 
 
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• 

• 

• 

census baiting; 

tracking techniques; 

census by live trapping; 

•  electronic methods of census. 

Pre-treatment activity measurement/estimation of numbers 

Indices of the target species population should be obtained both before and after the test 
treatment normally by at least 2 of the following quantitative methods. Other methods, 
such as electronic remote detection systems, can be used as additional information for 
example, in combination with bait census. 

Pre-treatment bait census 
The position of the census bait points should be indicated on the site sketch plan. Census 
bait should be laid for at least 4 days to cover the whole infestation in quantities at each 
bait point which as far as possible exceed the maximum daily take by rodents. The 
number of census baits should be approximately the same as the planned number of test 
bait points. Census points should not be located at the same place chosen to lay poison 
points but should be at different (intermediate) positions. Census bait should be different 
to the bait base used in the test product. 

The number of points where take has occurred and the amount of the take of the census 
bait, should be recorded daily. An indication of the change in weight of the bait due to 
moisture loss or uptake should be included. 

At the end of the bait census all baits and containers should be removed from the trial 
site. The total amount of census bait consumed will give an index of population size. 

Tracking activity measurement 
This is recommended for both rats and mice, and should be measured over at least 3 
days, simultaneously with the bait census, using tracking patches/boards laid around the 
site in numbers similar to the census bait points but as far as possible, not in the same 
locations. The locations of the patches/boards should be indicated on the plan. 

The patches/boards should be inspected for signs of activity and resurfaced daily. A 
simple scoring system can be devised to assess the number of rodent footprints per 
patch/board: summing the individual scores gives a daily activity index. When the pre-
treatment assessment is complete, the tracking patches/boards may be removed from 
the site or maintained to provide supplementary information on rodent activity. 

Census by trapping 
This is recommended for mice only, and should be carried out for a period of at least 3 
days using rodenticide-free bait in the live traps. Live traps should be laid around the 
site in numbers appropriate to the situation and likely population size. 

Animals caught should be marked by fur clipping and subsequently released. The 
numbers caught should be recorded and used to estimate the size of the population. 

The live traps should then be removed from the test site during the rodenticide 
treatment. 

Lag period 

Once the pre-treatment population measurement has been conducted there should be a 
lag period, normally 3-14 days (or longer for acute poisons where no pre-baiting is 
recommended) with no experimental interference (other than tracking) on the site. 

   
 
 
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Test treatment 

The test formulation must be applied in accordance with the draft SPC for an appropriate 
period (normally53 4 days for acute products and 30-40 days for multi-dose products). 
The locations of test bait points should, as far as possible, be different from those of the 
census bait points, traps, and tracking patches/boards. 

Where applicable the following items should be recorded: 

• 

• 

• 

the locations of the bait points on the plan; 

the amount of bait deposited at each point at each visit and the amount 
retrieved, including details of the type of container used; 

the number and species of rodents and other animals found dead, and the dates 
on which they were found; 

• 

the dates of all observations, treatments and censuses; 

•  any other information deemed relevant. This may include, for example weather 
conditions, temperature data, site changes instituted by the occupier (including 
improvements in hygiene and proofing), or supplementary information on rodent 
tracking activity. 

On termination of the treatment all poisoned baits and bait containers should be 
removed from the trial sites. Similarly rodent bodies should be searched for, removed 
and disposed of in the appropriate way for example, burial or burning. 

Post-treatment lag period 

On completion of the treatment there should be a lag period sufficient to allow poisoned 
animals to die or survivors to recover from the sub-lethal effects of the rodenticide. This 
period may be 3-14 days, depending on previous observations of time to death or full 
recovery. During this period there should be no experimental interference with the site 
other than tracking. 

Post-treatment activity measurement/estimation of numbers 

Once the post-treatment lag period is completed, the methods employed to measure 
pre-treatment activity should be conducted in exactly the same way. Traps, baits and 
tracking patches should be laid in exactly the same places as in the pre-treatment 
census. 

After each field trial, a comparison of population indices before and after treatment 
determines how successful the product has been in controlling the target population. The 
degree of control is expressed as a percentage reduction in the pre-treatment index. 

53 Deviation from this norm is possible but should be explained in the application. 

 
 
 
 
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Appendix 15. List  of  currently  available  standard  test 
methods for rodenticides 

This list may not be exhaustive, and makes no comment on the  suitability of particular 
test methods for efficacy testing. 

Table 38: List of standards 

Standard  

Title 

EPA/OPP Protocol 
Number 1.201 

Standard Norway Rat and Roof Rat 
Anticoagulant Liquid Bait Laboratory Test 
Method 

Target 
Organism(s)  

Mode of 
Application 

Brown 
Rat/Roof Rat 

Liquid bait 

EPA/OPP Protocol 
Number 1.202 

Standard House Mouse Anticoagulant Liquid 
Bait Laboratory Test Method 

House Mouse 

Liquid bait 

EPA/OPP Protocol 
Number 1.203 

Standard Norway Rat and Roof Rat 
Anticoagulant Dry Bait Laboratory Test 
Method 

EPA/OPP Protocol 
Number 1.204 

Standard House Mouse Anticoagulant Dry 
Bait Laboratory Test Method 

EPA/OPP Protocol 
Number 1.205 

Standard Norway Rat/Roof Rat 
Anticoagulant Tracking Powder Efficacy 
Laboratory Test Method 

EPA/OPP Protocol 
Number 1.212 

EPA/OPP Protocol 
Number 1.213 

Standard House Mouse Anticoagulant 
Tracking Powder Efficacy Laboratory Test 
Method 

Standard Norway Rat/Roof Rat 
Anticoagulant Wax Block and Wax Pellet 
Laboratory Test Method 

EPA/OPP Protocol 
Number 1.214 

Standard House Mouse Anticoagulant Wax 
Block and Wax Pellet Laboratory Test 
Method 

EPA/OPP Protocol 
Number 1.217 

Standard Norway Rat and Rood Rat 
Anticoagulant Placepack Laboratory Test 
Method 

EPA/OPP Protocol 
Number 1.218 

EPA/OPP Protocol 
Number 1.221 

EPA/OPP Protocol 
Number 1.225 

Standard House Mouse Anticoagulant 
Placepack Penetration Laboratory Test 
Method 

Proposed Norway Rat Anticoagulant 
Technical and Concentrated Dry Bait 
Laboratory Test Method 

Proposed House Mouse Anticoagulant 
Technical and Concentrated Dry Bait 
Laboratory Test Method 

Brown 
Rat/Roof Rat 

Dry Bait 

House Mouse 

Dry Bait 

Brown 
Rat/Roof Rat 

Tracking 
Powder 

House Mouse 

Brown 
Rat/Roof Rat 

Tracking 
Powder 

Wax Block 
and Wax 
Pellet 

House Mouse  Wax Block 

and Wax 
Pellet 

Brown 
Rat/Roof Rat 

Placepack dry 
bait 

House Mouse 

Placepack 
penetration 

Brown Rat 

House Mouse 

Technical and 
Concentrated 
Dry Bait 

Technical and 
Concentrated 
Dry Bait 

Liquid bait 

EPA/OPP Protocol 
Number: 1.207 

Standard Norway Rat/Roof Rat Acute Liquid 
Bait Laboratory test method 

Brown 
Rat/Roof Rat 

EPA/OPP Protocol 
Number: 1.208 

Standard House Mouse Acute Liquid Bait 
Laboratory Method 

House Mouse 

Liquid bait 

EPA/OPP Protocol 
Number: 1.209 

Standard Norway Rat/Roof Rat Acute Dry 
Bait Laboratory Test Method 

Brown 
Rat/Roof Rat 

Dry Bait 

EPA/OPP Protocol 
Number: 1.210 

Standard House Mouse Acute Dry Bait 
Laboratory Test Method 

EPA/OPP Protocol 
Number: 1.211 

Standard Norway Rat/Roof Rat Acute 
Tracking Powder Efficacy Laboratory Test 
Method 

House Mouse 

Dry Bait 

Brown 
Rat/Roof Rat 

Tracking 
Powder 

   
 
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Standard  

Title 

EPA/OPP Protocol 
Number: 1.219 

Standard Norway rat/Roof rat Acute 
Placepack Penetration Laboratory Test 
Method 

Target 
Organism(s)  

Mode of 
Application 

Brown 
Rat/Roof Rat 

Placepack 
penetration 

EPA/OPP Protocol 
Number: 1.220 

Standard House Mouse Acute Placepack Dry 
Bait Laboratory Test Method 

House Mouse 

EPA/OPP Protocol 
Number: 1.222 

Proposed Norway Rat Acute Technical and 
Concentrated Dry Bait Laboratory Test 
Method 

Norway rat 

EPA/OPP Protocol 
Number: 1.226 

Proposed House Mouse Acute Technical and 
Concentrated Dry Bait Laboratory Method 

House Mouse 

EPA/OPP Protocol 
Number: 1.227 

Proposed House Mouse Acute tracking 
Powder Efficacy Laboratory Method 

BBA 9 - 3.1 

Richtlinie für die Prufüng Prüfung von 
Nagetierbekämpfungsmitteln gegen 
Hausmause 

House Mouse 

House Mouse 

BBA 9- 3.2 

Richtlinie für die Prüfung von 
Nagetierbekämpfungsmitteln gegen 
Wanderratten 

Brown Rat 

EPPO 1982 

EPPO 1982 

EPPO 1986 

Guidelines for the Biological Evaluation of 
Rodenticides No1. Laboratory Tests for 
Evaluation of the Toxicity and Acceptability 
of Rodenticides and Rodenticide 
Preparations 

Guidelines For the Biological Evaluation of 
Rodenticides. Field Tests Against 
Synanthropic Rodents (Mus musculus, 
Rattus norvegicus, Rattus rattus) 

Guidelines for the Biological Evaluation of 
Rodenticides. Laboratory and Field Tests for 
the Evaluation of Rodenticidal Dusts 

- 

- 

- 

Placepack dry 
bait 

Technical  and 
Concentrated 
Dry Bait 

Technical  and 
Concentrated 
Dry Bait 

Tracking 
Powder 

Dry  and  liquid 
wax 
bait, 
block 
and 
pellets, 
contact 
rodenticides 

Dry  and  liquid 
wax 
bait, 
block 
and 
pellets, 
contact 
rodenticides 

- 

- 

- 

ASTM E 565-95 

ASTM E 593-95 

Standard Test Method for Efficacy of a 
Single-Dose Acute Rodenticide Under 
Laboratory Conditions for Commensal 
Rodents 

Standard Test Method for Efficacy of a 
Single-Dose Acute Rodenticide Under 
Laboratory Conditions 

Brown 
rat/Roof rat/ 
House mouse 

Brown 
rat/Roof rat/ 
House mouse 

Dry Bait 

Dry Bait 

EPPO 
Standards/97(2) 

Laboratory and field tests for the evaluation 
of rodenticidal dusts 

EPPO Standards 
/113(2) 

EPPO Standards 
/114(2) 

Laboratory tests for evaluation of the 
toxicity and acceptability of rodenticides and 
rodenticide preparations 

Field tests against synanthropic rodents 

- 

- 

Brown 
rat/Roof rat/ 
House mouse 

- 

- 

- 

 
 
 
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Title 

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Target 
Organism(s)  

Mode of 
Application 

EPPO Standards 
/169(2) 

Efficacy trials with rodenticide baits under 
practical conditions against Voles (Arvicola 
terestris and Microtus spp.) in their 
subterraean galleries" 

Voles 
(Microtus, 
Arvicola) 

EPPO Standards 
/197(1) 

Non-target effects of rodenticides 

EPPO Standards 
/198(1) 

Testing rodents for resistance to 
anticoagulant rodenticides 

- 

- 

- 

- 

- 

RRAC rat field 
trial protocol 
2013 

OECD 

Field Trial to Evaluate the Efficacy of 
Rodenticide Baits for the Control of Rats 
(Rattus norvegicus) 

OECD Guidance Document on the 
Recognition, Assessment and Use of Clinical 
Signs as Human Endpoints for Experimental 
Animals Used in Safety Evaluation (2002) 
http://www.oecd-
ilibrary.org/environment/guidance-
document-on-the-recognition-assessment-
and-use-of-clinical-signs-as-human-
endpoints-for-experimental-animals-used-
in-safety-evaluation_9789264078376-en 

Brown 
Rat/Roof Rat 

Dry Bait 

- 

- 

EPPO Standards 
PP1 2004 

2nd edition, volume 5, EPPO, Paris (2004), 
48-56. 

BBA (1963) 

BBA (1980) 

Méthode CEB 
n°254 (2013) 

Méthode 
n°257 (2014) 

CEB 

Richtlinie 9-2, Richtlinien für die Prüfung 
von Nagetierbekämpfungsmitteln gegen 
Schermaus (in German) 

Richtlinien für die amtliche Prüfung von 
Pflanzenbehandlungsmitteln 18-3.3, 
Richtlinie für die Prüfung von Rodentiziden 
gegen Schermaus im Forst (in German) 

Méthode d'essai d'efficacité pratique de 
générateurs de gaz fumigants pour lutter 
contre la taupe (Talpa europaea) et le 
campagnol terrestre (Arvicola terrestris) 
dans leurs galeries souterraines au champ. 

Méthode d'essai d'efficacité pratique 
d'appâts rodenticides pour lutter contre les 
campagnols (Arvicola terrestris, Microtus 
spp.) dans leurs galeries souterraines au 
champ 

Voles 

Voles 

Voles 

- 

- 

- 

Voles, moles 

Gassing agent 

Voles, moles 

Bait 

   
 
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Appendix 16.  Additional information on label claims 

NOTE to the reader:  

This  Appendix  contains  some  information  referring  to  the  Biocidal  Product  Directive 
that  is  now  obsolete.  This  will  be  revised  at  the  next  update,  but  in  the  meantime, 
readers  should  use  the  information  from  this  Appendix  in  conjunction  with  the 
information  available  in  Section  2  on  Claims  and  also  in  Section  5  in  the  general 
sections and under each PT section. The text in these sections has been revised and 
updated. 

Assessing the efficacy of biocidal products 

The evaluation of the efficacy of biocidal products differs greatly from that of active 
substances. 

Whilst the efficacy assessment of an active substance for Annex I inclusion requires only 
a minimal assessment, sufficient to show an innate level of activity for the active 
substance, the assessment needed for a biocidal product at the product authorisation 
stage is much more detailed. 

Rather than looking at innate effects, the efficacy assessment of a biocidal product is 
based on substantiating the efficacy claims made for a product.  The assessment is made 
on the product in its normal conditions of use. 

This principle is set out in paragraph 51 of Annex VI of the Directive (Common Principles 
for the Evaluation of Dossiers for Biocidal Products), which states: 

Data shall be submitted and evaluated to ascertain if the efficacy 

5.1 
claims of the biocidal product can be substantiated. Data submitted by the 
Applicant or held by the Member State must be able to demonstrate the efficacy 
of the biocidal product against the target organism when used normally in 
accordance with the conditions of authorisation. 

The label claims for the product must be submitted as part of the common core data set, 
as set out in Annex IIB (Common Core Data Set for Biocidal Products), which requires: 

V. INTENDED USES AND EFFICACY 

The proposed label claims for the product and efficacy data to 

5.10. 
support these claims, including any available standard protocols used, laboratory 
tests, or field trials, where appropriate 

As  the  label  claims  are  central  to  the  efficacy  evaluation  for  a  biocidal  product,  it  is 
important  to  understand  exactly  what  is  an  efficacy  claim,  and  be  able  to  identify  the 
individual components of a claim. 

Label claims for biocidal products 

As efficacy claims are assessed against the product ‘when used normally in accordance 
with the conditions of authorisation’, then it is important to define the ‘normal use’ of the 
product. 

There are several pieces of information which will form part of the conditions of 
authorisation which relate to the efficacy assessment.  These are: 

1.  The Formulation Type 

 
 
 
 
 
 
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This is determined by the product itself – e.g. a solvent based ready-for-use, a water 
based concentrate, a dusting powder, a gel bait, etc.  

2.  Application Method 

This is the method by which the product is intended to be applied. e.g. coarse spray, 
ultra-low volume (ULV) spray, bait station, skin lotion, etc. 

The application method may also describe a specific pattern of treatment.  This is 
particularly common for spray applications, but may also apply to other formulation 
types.  General descriptions of some common treatment patterns are given below. 

Surface treatments 

Crack and crevice treatments 

Contact (direct) spray treatments 

(i) 
These are treatments where the product is applied over surfaces such as walls, floors 
and ceilings, or as a treatment to outdoor surfaces. These treatments may involve 
treating a large area of surface or may only involve application to a narrow band. 
Surface treatments can also include application to temporary or permanent bodies of 
water (e.g. in mosquito control) and to solid and semi-solid manure. 
(ii) 
These are treatments where products are applied into cracks and crevices where 
insects hide and harbourage, or through which they may enter the building.  Such 
openings commonly occur at expansion joints, between different elements of 
construction and between equipment and floors. These openings may lead to voids 
such as hollow walls, equipment legs and bases, conduits and junction or switch 
boxes. 
(iii) 
These involve application directly onto insects, and are normally only possible when 
the insects are visible and available to be sprayed. 
In practice this often restricts direct application methods to controlling flying insects 
(such as adult moths and houseflies), although some limited control of minor 
infestations of crawling insects (such as ants or beetles) may be possible. 
(iv) 
These are treatments where the product is applied into the air rather than onto a 
surface. 
They are intended to disperse small droplets or particles into the atmosphere of a 
room or other open space, where they will normally stay for a period of time (very 
small particles may stay in the air for several hours under still conditions). 
(v) 
These are treatments where products are applied to limited areas on which insect 
pests are likely to occur, but which will not be in contact with food or utensils and will 
not ordinarily be contacted by workers.  These areas may occur on floors, walls and 
bases or undersides of equipment.  
(vi) 
Bait treatments use products that are intended to be ingested by the target.  This is 
normally through the insect feeding on the product directly, but may also include 
products which the target will come into contact with and later ingest during 
grooming/cleaning. 
The attractiveness of these products is through the use of a palatable food base, 
however they may also incorporate an attractant (e.g. a pheromone) which is 
intended to attract the target pests over a greater distance. 

Space treatments 

Spot treatments 

Baits 

3.  Application Rate 

This is the rate at which the product will be applied in use, e.g. apply 100 ml of product 
per square metre, apply at a rate of 1 bait station per 3 m2, spray for 20 seconds, etc. 

For efficacy assessment purposes, it is useful to consider the application rate as the 
amount of active substance applied to surface area or volume. 

   
 
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Unlike a human health or environmental risk assessment which look at the maximum 
amounts of product which are considered to be acceptable (i.e. if the amount of active or 
application rate increase, the risks to man or the environment will be unacceptable), an 
efficacy evaluation looks at the minimum application/dose rate which will be effective 
(i.e. if the application rate decreases, the product may not work). 

4.  Frequency of treatment and any specific interval between applications 

Some products will be used in a way that will require more than one treatment.  These 
products will give information on the treatment schedule which should be followed (e.g. 
insecticide re-treatment intervals or rodenticide re-baiting periods). 

Together, these pieces of information define the ‘normal use’ of the product (e.g. a 
solvent based ready-for-use product to be applied as a coarse spray at a rate of 
100 ml product m-2), and efficacy must be demonstrated for the product when it is used 
in this way. 

Whilst information on the application method and rate etc. will normally be clearly 
defined, the claims made for the effects of the product are much more difficult to 
identify. 

5.  Other specific conditions to be taken into account 

Occasionally, the “normal use” of a product will involve the use of the product in 
conjunction with other activities.  This will include the cleaning of an area prior to 
treatment.  The contributions made by other components of an Integrated Pest 
Management procedure may also have to be taken into account. 

Product labels and label claims 
The product label is the major source of information on a product.  It will give the use 
pattern to help determine the ‘normal use’ of the product, but will also make claims 
about the effectiveness of the product. 

These label claims form the core of any efficacy evaluation.  Efficacy is assessed mainly 
in relation to the claims made for the product. The norms and criteria set per insect pest 
will further guide the evaluation.   

Whilst the phrase ‘label claims’ is generally used, this phrase actually encompasses all 
claims made for the product, not just those made on the label itself.  Claims may also be 
made for a product with any accompanying information (such as leaflets) or on 
advertising material. 

For efficacy purposes, all of these claims also have to be justified before they can be 
allowed onto a label. 

What is a label claim? 
A label claim is anything on the product label that makes a claim about what the product 
does or the benefits that will result from its use. At this moment there is no standard 
format for making claims about the effects and benefits of using the product, and the 
type and style of label claims can vary widely between different Member States. 

For example, a product which claims to be ‘For the control of cockroaches’ in one 
Member State may claim that it ‘Kills cockroaches fast!!’ in another. 

To aid in the evaluation process, a standardised method for identifying the main 
components of a label claim is set out below. 

Label claims – understanding the components 
A set of label claims will consist of 2 types of information which describe what the 
product will do when it is used (in accordance with its ‘normal use’). These are: 

 
 
 
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1.  The target species which the product will be effective against 

and 

2.  The effect (or effects) which the use of the product will have on the target species 

and the benefits which may result from this effect 

Target species 

The product label will give details about which species the product is to be used against.  
This information will often be quite specific (e.g. ‘for the control of pharaohs ants’ or ‘kills 
ants, cockroaches, fleas and bed bugs or repels mosquitoes’).  In these cases it is easy 
to identify what are the target species. 

However there can also be instances where a more general claim is made, such as for 
use against ‘crawling insects’.  In these cases, it is difficult to require data on every 
crawling insect. 

They will need to supply efficacy data on relevant representative species, which may be 
those used in standard test methods or those that the Applicant argues are 
representative of the use pattern of the biocide and the nature of the application (e.g. 
whether it is a space application or a surface application). 

In some instances it is possible to allow a compromise on the label. For example, 
members of the general public may not know what species of fly is in their home, but 
the regulators will need to know what the product is effective against. In this particular 
instance it may be possible to allow a claim such as ‘Effective against flying insects such 
as the housefly, mosquitoes and midges’. 

The effects of using the product 

The remaining parts of the label claim will describe the effects on the target organisms 
and benefits of using the product. 

The major effects which are generally claimed are that a product will: 

•  kill, knock down, repel, attract, reduce the numbers of or inhibit a target 

organism 

•  control, reduce or prevent the build-up of a population 

•  prevent or reduce an undesirable effect. 

For insecticide products, the following claims are the ones that are frequently 
encountered: 

‘Kill’ claims generally refer to the death of an individual or a number of individuals (the 
death of an entire population is more generally found under a ‘control’ claim) and 
generally refer to an existing infestation. 

‘Knockdown’ claims are generally restricted to insecticides and acaricides.  A knockdown 
effect is one where a target insect becomes unable to carry out coordinated movement, 
but has not been killed. 

Knockdown effects are often included in an insecticide product to produce a rapid, visible 
effect on a target in order to satisfy user expectations.  These effects can be reversible, 
with insects able to recover after a period of time. Recovery is often dependent upon 
dose administered. 

Knockdown claims may be found in conjunction with a kill claim, and many ‘dual action’ 
insecticide products contain two active substances - with one active substance producing 
a quick knockdown effect (such as a flying insect falling out of the air) whilst a second, 
slower acting, active substance produces the killing effect.  Combined claims may be 
along the lines of ‘knockdown within 10 minutes and kill within 2 hours’. 

   
 
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When it comes to efficacy testing, some companies use the two terms interchangeably, 
so you will get products or test reports mentioning ‘knockdown’ where a killing effect is 
actually meant. For evaluation purposes, knockdown and kill are considered to be 
separate effects. 

‘Complete control’, ‘colony kill’ or ‘nest kill’ claims will generally refer to the elimination 
of an entire infestation or population - i.e. use of the product will essentially ‘remove the 
problem’. 

As stated above, the mortality of individuals (rather than populations) is considered to 
be a ‘kill’ effect. 

To highlight the difference between ‘kill’ and ‘control’, we can take the example of an ant 
nest outside of a house, close to the back door. The queen (which does all of the 
reproduction) remains hidden away in the nest and produces new ants for the colony, 
and the only ants seen outside of the nest are the sterile female workers. 

An aerosol product which is intended to be sprayed onto ants wandering around in your 
kitchen to kill them will only be having a ‘kill’ effect.  Killing off individuals or numbers of 
workers will have little effect on the nest and the colony as a whole, as the queen and 
fertile males will remain unaffected in the nest. 

In order to remove the problem, you actually have to kill off the colony.  So a product 
claiming to ‘control’ an infestation of ants would have to eliminate the queen or disrupt 
the ability of the colony to reproduce. 

‘Reduce’ claims will generally refer to reducing the numbers of (but not completely 
eliminating) a target population. Whilst not eliminating an infestation may seem to be an 
odd claim to make, there are situations where it would be practically impossible to totally 
control a target population and where the best result is to reduce the scale of the 
problem. 

An example of this would be reducing the fly burden in a poultry house or intensive 
animal house. However, the issue of resistance must always be kept in mind when 
considering treatments which do not fully control a population. 

More complex label claims 

Whilst  a  label  claim  is,  at  its  most  basic,  a  target  and  an  effect,  most  claims  are  more 
complex,  introducing  further  elements  beyond  the  basic  target/effect  combination 
described above. 

These  additional  parts  of  a  label  claim  more  fully  describe  the  effects  on  the  target 
organisms and benefits to be gained from using the product. 

Claims  for  the  effects  and  benefits  of  using  the  product  can  generally  be  broken  down 
into 6 major components, which are described in Table 26. 

The  examples  given  in  the  table  cannot  be  exhaustive,  but  are  given  to  illustrate  the 
type of information which appears in label claims. 

Table 39: Components Making Up a Label Claim 

Group 

Label Claim 

A 

B 

Against what target organism(s) will the product be used? 

Target 
organism(s) 

•  Specific insect (e.g. ants) 
•  Several insects (e.g. ants and wasps) 
•  General claim (e.g. flying and crawling insects) 

Type of effect  What effect will the use of the product have on the target? 

 
 
 
 
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Group 

Label Claim 

Examples include: 

•  Kill 
•  Knockdown 
•  Control 
•  Flushing 
•  Attracting 
•  Repelling 

C 

D 

E 

F 

G 

How long will the product take to produce the effect? 

Time taken 
to produce 
the effect 

Examples include: 

•  within 5 minutes 
•  within 1 hour 
•  within 3 months 

In what types of environment and on what type of surfaces will 
the product be used? 

For example: 

Area of use 

indoors/outdoors 

• 
•  on hard porous and non-porous surfaces 
•  on soft furnishings 
• 
• 

in hospitals 
in and around buildings 

Will the product have a residual effect, and if so, how long for? 

Duration of 
the effect 

For example: 

• 
• 

for 6 weeks 
for 3 months 

User 

Who can use the product? 

Industrial use 
• 
•  Professionals 
•  Consumers 

Does the product claim any other specific benefits? 

Other specific 
claims 

Examples include: 

•  works against resistant species 
•  helps prevent biting 
•  protects fabric from damage 

A label claim will not always contain all 7 components.  For example, where no residual 
activity is being claimed, section E will not be represented, and where no specific other 
claims are being made, claims in section G will not be present. 

The target organism (A), the type of effect (B) and area of use (D) and the user (F) 
should always be given. 

On some labels, the time taken to product the effect (C) will not have been given (e.g. 
‘for the control of cockroaches’) or is not a specific value (e.g. ‘kills flies fast’). In these 

   
 
 
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cases, the evaluator will use the norms and criteria given per insect for the evaluation of 
the data. 

Linking the components of the label claim 

When initially trying to understand how the components of the label claims fit together, 
it can help to place the assorted claims into a table in order to identify how the various 
elements interact. For example: 

Table 40: Example of linking label claims 

Label claim (B) 

Effect time (C)  Area of use (D) 

Duration of effect (E) 

Knocks down 

within 5 minutes 

-  on  hard  porous  and 
non-porous surfaces 

for 6 weeks 

Kills 

within 1 hour 

- on soft furnishings 

The beneficial effect of the product (B) will be accompanied by the timescale in which the 
effect will happen (C).  In these cases, it must be demonstrated that the product will be 
efficacious within the stated time. 

In the above example, it must be demonstrated that the product is capable of both 
knocking down the target insects within 5 minutes AND killing them within 1 hour. 

The area of use (D) gives information about the conditions in which the product will be 
used and the type of surfaces it will be used on. The efficacy data supplied should 
demonstrate that the product will be efficacious in the areas specified or on 
representative surfaces of the types described. 

In the example, it would have to be demonstrated that the product would produce its 
knockdown and kill effects within the times stated AND on both hard surfaces and soft 
furnishings. 

The duration of effect (E) specifies the length of residual activity which must be 
demonstrated. 

In the example, it must be demonstrated that the product is still capable of producing 
the effects on the specified surfaces 6 weeks after treatment (although not necessarily to 
the same degree as a fresh treatment). 

Other claims can be linked into this process in the same way.  For example, if claims 
were being made that the product was to be used against resistant individuals, then all 
of the above elements would have to be proved using a resistant test population to 
generate the data. 

Once the various elements making up the label claims have been identified then the 
evaluation of the efficacy data submitted can proceed. 

General guidance on the assessment of label claims is included in the paper “Broad 
principles of assessing efficacy in relation to claims made on the label for biocidal 
products”, which was agreed at the Technical Meeting TM III 05 in October 2005, and at 
the subsequent CA meeting. 

Guidance on type of and amount of data which would normally be required to support 
many of the major label claims is given for the main pest species elsewhere in this 
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Appendix 17. Species grid 

Table 41: PT 18 Crawling Insects 

Action 

SITE  APPLICATION 

CLAIM 

TEST SPECIES 

RATIONALE 

NOTES 

1A 

Flushing 

Indoor  Crack & Crevice 

METHOD 

“Flushes 
cockroaches out of 
hidden places” 

Blattella germanica or 
Periplaneta 

1B 

Knockdown 

Indoor  Direct Spray 

“Knocks down 
cockroaches”; 
“Knocks down 
cockroaches in x 
seconds” 

Blattella germanica and 
either  
Periplaneta 
species or 
Blatta orientalis  

INSECT 
STAGE 

Data show Periplaneta 
flush before Blattella 
N.B This is true with 
pyrethroids, the case 
may be different with 
other actives. Fast acting 
pyrethroids may 
knockdown Blattella 
faster than they can be 
flushed, use Periplaneta 
in this case. 

These species are 
representative of all 
domestic cockroaches 
found in Europe and 
around the world. 
Behavioural differences 
between species do not 
come into play when 
testing aerosols for direct 
spray efficacy. 

Any additional species need 
specific data. 

Nymphs 
Adults 

Adults 

We see little or no value in 
producing nymph/immature 
data in aerosol direct spray 
tests. Testing with only adults 
provides a very clear picture of 
product activity for registration 
studies. More than one life 
stage is an unnecessary 
burden. 

1C 

Kills 

Indoor  Direct Spray 

“Kills cockroaches”; 
“Kills cockroaches in 
x seconds” 

Blattella germanica and 
either Periplaneta 
species or 
Blatta orientalis 

1D 

Kills 

Indoor  Direct Spray 

“Kills ants”; 
“Kills in x seconds” 

Lasius sp. 

1E 

Kills 

Outdo
or 

Direct Spray 

“Kills ants”; 
“Kills in x seconds” 

Lasius sp. 

See B. 

See B 

Adults 

Monomorium ants are 
much smaller and more 
sensitive so would be 
covered by data for 
Lasius 

Adults 

Adults 

   
 
 
 
 
 
 
 
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Action 

SITE  APPLICATION 

CLAIM 

TEST SPECIES 

RATIONALE 

NOTES 

1F 

Knockdown 

Indoor  Direct Spray 

METHOD 

“Kills crawling 
insects and other 
arthropods” 

1G 

Knockdown 

Indoor  Space spray; 

aerosols,  gases, 
fogs, smokes 

Knocks down 
crawling insects 

See B 

Multiple species are 
common world-wide. Test 
species will depend upon 
seasonal and local 
availability. 

See also B.  

C + D and a variety of 
other common species 
e.g. Forficula 
auricularia, Acheta 
domesticus, Cimex 
lectularius, Attagenus, 
Dermestes sp., fleas, 
silverfish, booklice, 
carpet beetles, 
woodlice, ticks, 
centipedes, spiders 

Wood borers, carpet 
beetles, stored product 
beetles, other small 
crawling insects. 
Data required for claims 
on cockroaches (C) and 
fleas as surrogates for 
others 

1H 

Kills 

Indoor  Space spray; 

Kills crawling insects  Wood borers, carpet 

aerosols,  gases, 
fogs, smokes 

1I 

Residual 
Kill 

Indoor  Surface or Crack 
& Crevice Spray, 
Powders 

“Kills cockroaches”; 
“Kills cockroaches 
up to x weeks or 
months” 

beetles, stored product 
beetles, other small 
crawling insects. 
Data required for claims 
on cockroaches (3) and 
fleas 

Blattella germanica and 
either  
Periplaneta 
species or 
Blatta orientalis 

Consider substrate and ageing 
period in the method 

435 

INSECT 
STAGE 

Adults 

Adults, 
immatures 

Adults, 
immatures 
and if 
claimed eggs 

Adults and or 
immature 
stages. 
Specify 
realistic 
exposure 
period 
followed by 
reasonable 
“recovery” 
period.  

 
 
 
 
 
 
 
 
 
 
 
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Action 

SITE  APPLICATION 

CLAIM 

TEST SPECIES 

RATIONALE 

NOTES 

METHOD 

1J 

Residual 

Indoor  Surface or Crack 
& Crevice Spray, 
Powder 

Kills ants”; 
“Kills ants for x 
weeks or months” 

Lasius sp. and/or 
Monomorium pharaonis 
as option 
(see 4) 

Consider substrate and ageing 
period in the method 

1K 

Residual 

Indoor  Surface or Crack 
& Crevice Spray, 
Powder 

“Kills crawling 
insects and 
arthropods” ; “Kills 
for x weeks or 
months” 

K + L and a variety of 
other common species 
e.g. Forficula 
auricularia, Acheta 
domesticus, Cimex 
lectularius, Attagenus, 
Dermestes sp., fleas, 
silverfish, booklice, 
carpet beetles, 
woodlice, ticks, 
centipedes, spiders 

We propose only roaches be 
tested for full period. 

1L 

Residual 

Indoor  Bait 

“Kills cockroaches”; 
“Kills cockroaches 
for x weeks or 
months”;   

Blattella germanica; 
Periplaneta americana 
and Blatta orientalis 

Either the claim is limited to a 
specific species or the three 
species are tested 

INSECT 
STAGE 

Adults 
Specify 
realistic 
exposure 
period 
followed by 
reasonable 
“recovery” 

Adults and 
immature 
stages. 
Consider 
substrate 
and ageing 
period in 
method.  
Specify 
realistic 
exposure 
period 
followed by 
“reasonable”  
recovery 
period. 

Nymphs 
Adults. 
Consider 
ageing period 
in method. 
Provide 
harbourage 
and 
alternative 
food and 
water. 

1M  Secondary 
kill 

Indoor  Bait 

“Kills cockroaches 
that do not visit the 
bait (secondary 

Blattella germanica; 
Periplaneta americana 
and Blatta orientalis 

Life stage to be tested 
depends upon a specific 
mode of action 

Either the claim is limited to a 
specific species or the three 
species are tested 

Life stage to 
be tested 
depends 

   
 
 
 
 
 
 
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Action 

SITE  APPLICATION 

CLAIM 

TEST SPECIES 

RATIONALE 

NOTES 

METHOD 

kill)” 

(necrophagy versus 
coprophagy).  Either 
nymphs or adults could 
be used. 

437 

INSECT 
STAGE 

upon a 
specific mode 
of action 
(necrophagy 
versus 
coprophagy). 
Either 
nymphs or 
adults could 
be used. 

1N  Nest kill 

Indoor  Bait 

1O 

Kill 

Indoor  Bait 

control of entire 
population of 
cockroaches 

Blattella germanica; 
Periplaneta americana 
and Blatta orientalis 

“Kills ants”; “Kills 
ants for x weeks or 
months”; 

Monomorium pharaonis 
and /or Lasius niger. 

1P 

Colony kill 

Indoor  Bait 

“Kills the queen and 
the colony” 

Monomorium pharaonis 
and /or Lasius niger. 

1Q 

Kills 

Indoor  Spray, powder 

“Kills dust mites” 

Dermatophagoides sp. 

1R 

Residual 
Kill 

Indoor  Spray, powder 

“Kills dust mites for 
x weeks/months” 

Dermatophagoides sp. 

Either the claim is limited to a 
specific species or the three 
species are tested 

Nymphs 
Adults 

Either the claim is limited to a 
specific species or the two 
species are tested. 
Provide harbourage and 
alternative food and water. 

Either the claim is limited to a 
specific species or the two 
species are tested. 
Provide harbourage and 
alternative food and water. 

Adults and all 
immature 
stages 

Adults and all 
immature 
stages. Use 
entire 
colonies 
including 
queens.  

Adults and all 
immature 
stages, if 
claim include 
eggs. 

Consider substrate and ageing 
period in method.  Specify 
realistic insect exposure period 
followed by reasonable 
“recovery” period.  

Adults and all 
immature 
stages, if 
claim include 
eggs. 

 
 
 
 
 
 
 
 
 
 
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1S 

Kill 

1T 

Kill 

1U 

Kill 

1V 

Kill 

Action 

SITE  APPLICATION 

CLAIM 

TEST SPECIES 

RATIONALE 

NOTES 

INSECT 
STAGE 

METHOD 

Outdo
or 

Baits, Dusts, 
powders 

Kills ants 

Lasius sp. 

Outdo
or 

Baits, Dusts, 
powders 

“Kills the queen and 
the colony” 

Lasius sp. and /or 
Monomorium pharaonis  

Either the claim is limited to a 
specific species or the two 
species are tested. 
Provide harbourage and 
alternative food and water. 

Outdo
or 

Sprays, liquid 
drenches 

Kills ants 

Lasius sp. 

Add colony kill 

Outdo
or 

Sprays, liquid 
drenches 

“Kills the queen and 
the colony” 

Monomorium pharaonis 
and /or Lasius niger. 

Either the claim is limited to a 
specific species or the two 
species are tested. 
Provide harbourage and 
alternative food and water. 

Whole colony 

1W  Kill or 

repellent 

Outdo
or 

1X 

Kill or 
repellent 

Outdo
or 

Physico-
chemical 
barrier. 
Installation 
between the soil 
and the future 
construction 

Chemical 
barrier 
Injection in wall 
and soil  

Preventive 
Pre- construction 
treatment 
Prevent construction 
attack 

All subterranean 
termites Reticulitermes 
sp. 
Coptotermes sp. 
Heterotermes sp.  

Preventive 
Pre-construction 
treatment 
Prevent construction 
attack 

All subterranean 
termites Reticulitermes 
sp. 
Coptotermes sp. 
Heterotermes sp. 

1Y 

Kill or 
repellent 

Outdo
or 

Chemical 
barrier 
Injection in wall 
and soil 

Curative 
Post-construction 
treatment 

All subterranean 
termites Reticulitermes 
sp. 
Coptotermes sp. 
Heterotermes sp. 

1Z 

Kill 

Outdo
or 

Baits system 

Curative 
Post-construction 
treatment 

Reticulitermes sp. 
Coptotermes sp. 

Due to the specificity of baits, 
only species tested should be 
claimed on the product label 

   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Action 

SITE  APPLICATION 

CLAIM 

TEST SPECIES 

RATIONALE 

NOTES 

METHOD 

1AA  Kill  

Indoor  Curative  

(Prevention is PT 
8) 

Colony elimination 

Kills dry wood 
termites 

1AB  Barrier 

treatment 

Indoor 
/ 
Outdo
or 

Sprays, Powders  Prevents entry of 

crawling insects for 
x weeks or months 

e.g. Cryptotermes sp. 

Blattella germanica and 
either Periplaneta 
species or B. orientalis, 
Lasius sp. 
See list above (“F”) for 
selection, but expect 
roaches and ants to be 
the main claim 

Table 42: PT 18 Flying Insects 

Action 

SITE 

APPLICATION 
METHOD 

CLAIM 

TEST SPECIES 

RATIONALE 

NOTES 

439 

INSECT 
STAGE 

INSECT 
STAGE 

adults 

2A 

Kills/ 
Knocks 
down 

2B 

kills 

2C 

2D 

2E 

Indoor 

Direct spray or 
room treatment 

Indoor/ 
Outdoor 

Aerosol, Coils, 
mats or liquid 
electrics; 
Plaquettes or 
similar devices 

“Knocks down 
and/or Kills flies, 
mosquitoes”; 

Kills mosquitoes  
for up to x hours  

Outdoor   Nuisance flying 

Kills “XYZ” 

insects 
( landfill area)  

Outdoors  Direct and 

Kills “XYZ” 

residual sprays 

Indoor  

Fumigants 

Kills “XYZ” 

Musca domestica; Culex 
sp. or Aedes sp.  

These two species are 
representative of most 
urban species. 

Flies and mosquitoes would 
be proxy insects for gnats 
and midges 

Culex sp. or Aedes sp. 

All insects, for which claims 
are made, should be tested. 

adults 

Musca domestica 
Culex sp. or Aedes sp. 

Claimed insects need to 
be tested 

Claimed insects need to 
be tested 

All insects, for which claims 
are made, should be tested. 

adults 

adult and 
larvae 

All insects and insect stages 
for which claims are made, 
should be tested. 

Adults , 
eggs, and 
larvae 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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440 

Action 

SITE 

2F 

kills 

Indoor 

2G 

kills 

Indoor 
Outdoor 

2H 

kills 

Outdoor 

2I 

kills 

Indoor 

APPLICATION 
METHOD 

Direct spray or 
room treatment 

CLAIM 

TEST SPECIES 

RATIONALE 

NOTES 

“Kills flying moths”  Plodia interpunctella or 

Tineola bisselliella 

/ 

Direct spray  

“Kills wasps” 

 Vespula sp. 

Nest treatment 
(all methods) 

“Kills wasp nests”; 
“Kills the queen” 

 Vespula sp. or 
Dolichovespula sp. 

Test on whole nests 

Closet or 
confined space 
treatments 

“Kills clothes 
moths and 
larvae”;  “Kills for 
x weeks or 
months” 

Tineola bisselliella 

All insects, for which claims 
are made, should be tested. 

2J 

kills 

Indoor  

Baits  

Kills “XYZ”flies  

2K 

kills 

Outdoor   Mosquitoes 

Kills mosquito 
larvae 

Specifc species claimed 
on the label 

Culex sp. Or Aedes sp. 

2L 

kills 

Indoor 
Outdoor 

/ 

Fly larvicides 

Kills “XYZ”flies 

Specifc species claimed 
on the label 

for IGRs the larval stage 
needs to be selected 
according to the mode of 
action. 

for IGRs the larval stage 
needs to be selected 
according to the mode of 
action. Specify substrates)  

last instar 
larvae 

INSECT 
STAGE 

adults 

adults 

adults, 
queen for 
specific 
claim 

adults, 
eggs and / 
or larvae 
depending 
upon claim 

adults 

last instar 
larvae 

   
 
 
 
 
 
 
 
 
 
 
 
 
 
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441 

Appendix 18. List  of  currently  available  standard  test  methods  for  product  type  18 
insecticides/acaricides  and  product  type  19  repellents/attractants  (as  far  as  they 
concern insects and other arthropods) 

Recognised  standard  methods  for  the  efficacy  testing  of  biocidal  products  intended  for  the  control  of  insects,  acaricides  and  other 
arthropods.  

This  is  a  non-exhaustive  list  of  available  standard  methods  without  distinction  on  suitability,  usefulness,  repeatability,  the  order  of 
acceptability or robustness. 

 
 
 
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442 

Table 43: General 

Reference 

Title 

PT 

Short description 

Reference 
source 

US EPA 

OPPTS 810.3000 

CEB 196 

General Considerations for 
Efficacy of Invertebrate Control 
Agents 

18  General guide 

18 

Test method to evaluate the efficacy of insecticidal bait 
products against common ant species 

Vegephyl 

EPPO pp1/152 

Design and analysis of efficacy 
evaluation trials 

18 

EPPO pp1/181 

Conduct and reporting of 
efficacy evaluation trials, 
including good experimental 
practice 

18 

EPPO Bulletin Volume 
18, Issue 2, 1988  
(p. 337-341)  

EPPO Recommendations on 
fumigation standards 

18 

OPPTS 810.3200 

Livestock, poultry, fur- and 
wool-bearing animal treatments 

18 

This standard provides detailed advice on the design and 
analysis of efficacy evaluation trials. Primarily intended for 
use in plant protection but also very useful for biocides. 

This standard provides guidance on how to organize trials, 
and how to plan, conduct and assess them, then record 
and interpret them, so as to obtain comparable and 
reliable results. It is also based on the principle that trials 
should be performed according to Good Experimental 
Practice (GEP). 

This Product Performance Test Guidelines concerns 
efficacy testing of invertebrate control pesticides used on 
cattle, horses, sheep, goats, swine, chicken, turkeys, 
other domestic fowls, and fur-bearing animals, such as 
mink and rabbits. 

EPPO 

EPPO 

EPPO 

US EPA 

OPPTS 810.3300  

Treatments to control pests of 
humans and pets 

18 

This guideline is concerned with efficacy testing of 
invertebrate control pesticides used on humans and pets. 

US EPA 

OPPTS 810.3500 

Premises treatments 

18  This guideline provides recommendations for the design 

US EPA 

   
 
 
 
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Reference 

Title 

PT 

Short description 

SANS 5233 

SANS 5576 

SANS 5583 

SANS 6136 

SANS 5689 

SANS 5690 

and execution of laboratory and field studies to evaluate 
the performance of products applied in or around 
premises. 

Pesticides - Biological 
evaluation of mists and fogs 

18 

This standard specifies a method for the biological 
evaluation of the efficacy of pesticidal mists and fogs. 

Pesticides – Biological 
evaluation of insecticidal oil-
based space spray in low-
pressurized dispensers 

Pesticides – Biological 
evaluation of the contact 
efficacy of liquid residual 
insecticides 

Pesticides – Biological 
evaluation of materials that 
release an insecticide upon 
heating 

Pesticides – Biological 
evaluation of the direct spray 
knockdown and killing 
proprieties of liquids and 
aerosol dispensers 

Pesticides - Biological 
evaluation of the properties of 
solid fly baits 

18 

18 

18 

This standard specifies a method for the determination of 
insecticidal oil-based space sprays in low-pressurized 
dispensers. 

This standard describes a method for conducting biological 
contact efficacy tests of liquid residual insecticides 
(including wettable powders and water dispersible 
granules). 

This standard describes the method for conducting 
biological efficacy tests on materials that release 
insecticides on heating. 

18 

This standard describes a method for conducting biological 
evaluation tests of liquid and aerosol formulations. 

SABS 

18 

This standard describes a method for conducting biological 
evaluation tests on solid fly baits. 

SABS 

SANS 5807 

Rooms, conditions, equipment,  18  This standard specifies the test rooms, test conditions, 

SABS 

443 

Reference 
source 

SABS 

SABS 

SABS 

SABS 

 
 
 
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Reference 

Title 

PT 

Short description 

Reference 
source 

and insects and their handling, 
for use in pesticide testing 

test equipment, test insects and their handling, for use in 
pesticide testing. 

SANS 899 

Pesticides - Insecticidal space 
spray in pressurized aerosol 
dispensers 

CTD/WHOPES/IC/96.1 

Evaluation and testing of 
insecticides 

18 

18 

This standard covers requirements for insecticidal space 
sprays in pressurized aerosol dispensers and is intended 
for use in food-handling, food-processing and catering 
establishments. 

Report of the WHO Informal Consultation on the 
evaluation and testing of insecticides, WHO, Geneva, 7-11 
October 1996 

SABS 

WHO 

Table 44: Crawling Insects: Cockroaches 

Reference 

Title 

PT 

Short description 

Reference 
source 

CEB 249 

CEB 159 

18 

18 

Method for testing the efficacy in the laboratory and under 
practical conditions of use, of insecticidal baits intended 
for the control of cockroaches in premises 

Vegephyl 

Trial method to evaluate the efficacy of insecticidal 
products for the control of cockroaches in buildings under 
practical conditions 

Vegephyl 

ENV/JM/MONO(2013)3 

SANS 5458 

Guidance Document on Assays 
for Testing the Efficacy of Baits 
against Cockroaches  

Pesticides – Rearing and 
handling of the German 
cockroach (Blatella germanica 

18 

Outlines methods available for testing efficacy and 
effectiveness of baits against cockroaches. 

18 

This standard specifies a method for the rearing and 
handling of the German cockroach (B. germanica (L.). 

OECD 

SABS 

   
 
 
 
 
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Version 4.0 December 2021 

Reference 

Title 

PT 

Short description 

(L.)) 

445 

Reference 
source 

WHO/VBC/75.593 

Instructions for determining the 
susceptibility or resistance of 
cockroaches to insecticides 

18 

The purpose of this test is to detect the presence of 
resistant individuals in a mosquito larval population. 

WHO 

Table 45: Crawling Insects: Termites 

Reference 

Title 

Evaluation of the anti-termite 
efficacy of a barrier placed in 
an alkaline medium.  

Evaluation of the anti-termite 
efficacy of a physico-chemical 
barrier - Field test - Device 
without concrete slab. 

Exposure of anti-termite 
physico-chemical barriers to 
solar radiation. 

CTBA-BIO-E-007 

CTBA-BIO-E-008/2 

CTBA-BIO-E-016 

FCBA-BIO-E-041 

FCBA-BIO-E-045 

PT 

18 

18 

18 

Short test description 

Reference 
source 

Laboratory test method to assess the loss of efficacy that 
an alkaline medium could induce on an anti-termite 
barrier. 

This test method presents the different steps to evaluate 
the effectiveness of an anti-termite protective film in an 
outdoor environment. 

This test method presents the principles that must be 
applied to expose plastic materials to solar radiation. It is 
applicable to plastic-based physico-chemical termite 
barriers. 

18 

Efficacy criteria for performance of CTBA-BIO-E-xx and 
FCBA-BIO-E-xx tests 

18 

This method is designed to test the preventive treatment 
of  physico-chemical  barrier  with  a  concrete  slab.  All  the 
efficacy  tests  performed  with  CTBA-BIO-E-008  remain 
valid. 

FCBA 

FCBA 

FCBA 

FCBA 

FCBA 

 
 
 
 
 
 
446 

Reference 

Title 

FCBA-BIO-E-053 

NF X 41-542 

NF X 41-543-1 

Wood preservatives - Anti-
termite treatment product for 
floors, walls, foundations and 
masonry - Accelerated aging 
test of treated materials before 
biological tests - Percolation 
test. 

Wood preservatives - 
determination of the efficacy of 
a bait-trap system - part 1: 
Efficacy of the insecticide 
formulation - laboratory 
method.  

Guidance on the BPR: Volume II Parts B+C  
Version 4.0 December 2021 

PT 

18 

Short test description 

This method replaced CTBA-BIO-E-001 and CTBA-BIO-E-
002. All the efficacy tests performed following CTBA-BIO-
E-001 and CTBA-BIO-E-002 methodologies remain valid. 

Reference 
source 

FCBA 

8+
18 

Laboratory test method to determine the efficacy against 
termites of products or material used as barrier designed 
for ground and/or wall. 

AFNOR 

8+
18 

This test method is applicable to sustained insecticidal 
formulations intended for use in bait trap systems. 

AFNOR 

NF X 41-543-2 

Wood preservatives - 
determination of the efficacy of 
a bait-trap system - part 2: 
field method.  

8+
18 

This test method is intended to evaluate the efficacy of 
the baits in an experimental site where termite activity is 
reported. Consumption of the tested bait must be 
registered at least in the first 6 months after the 
introduction of the baits. The elimination of termites in the 
experimental site should be registered maximum after 18 
months (counted since the introduction of the first tested 
bait), excluding the winter period.  

AFNOR 

NF X 41-543-3 

Wood preservatives - 
determination of the efficacy of 
a bait-trap system - part 3: 
performance criteria.  

8+
18 

Applies  to  anti-termite  products  with  a  delayed  effect 
intended for use in an identified bait trap system and the 
methods of use of which are known. It defines the criteria 
to be achieved in the tests described in part 1 and part 2. 

AFNOR 

   
 
 
 
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Reference 

Title 

PT 

Short test description 

NF X 41-550 

NF X 41-551 

Termites - Determination of the 
effectiveness against termites 
of products or materials used 
as barrier designed for ground 
and/or wall - Laboratory 
method 

Termites - Determination of the 
effectiveness against termites 
of products or material used as 
barrier designed for ground 
and/or wall- Performance 
criteria 

8+
18 

8+
18 

It describes a test method for determining the 
effectiveness of a product or material intended to 
constitute a protective barrier for constructions against 
subterranean termites. To characterize the persistence of 
the efficacy of these products and materials, this method 
can be applied following an aging test. 

It defines the criteria for the effectiveness of products or 
materials intended to constitute a protective barrier for 
buildings against subterranean termites.  

447 

Reference 
source 

AFNOR 

AFNOR 

OPPTS 810.3800 

Methods for efficacy testing of 
termite baits 

8+
18 

This test method concerns the product performance 
testing for evaluation of products used as baits to kill and 
control termites. 

US EPA 

Table 46: Crawling Insects: Other Crawling Insects 

Reference 

Title 

PT 

Short test description 

AATCC 194 

Test Method for Anti-House Dust 
Mite Properties of Textiles under 
Long-Term Test Conditions 

18 

This test method is for the evaluation of the degree of 
anti-house dust mite activity in a long-term testing 
environment for textiles treated at the manufacturing 
level for this purpose. 

Reference 
source 

AATCC 

OCSPP 810.3900 

Product Performance Test 
Guidelines; OCSPP 810.3900 
Laboratory Product Performance 

PT 
18 

This guideline provides recommendations for the design 
and execution of laboratory test to evaluate the 
performance of products intended to repel, attract, and/or 

US EPA 

 
 
 
 
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Reference 

Title 

PT 

Short test description 

Reference 
source 

Testing Methods for Bed Bug 
Pesticide Products 

kill the common bed bug (Cimex lectularius). 

OPPTS 810.3100 

Soil treatments for imported fire 
ants 

18 

This guideline contains recommended test methods for 
evaluating the performance of products for the treatment 
and control of imported fire ants. 

US EPA 

Table 47: Flying Insects  

Reference 

Title 

PT 

Short test description 

Reference 
source 

CEB 107 

Method for testing the practical 
effectiveness of insecticidal 
products intended for the control 
of barn flies in premises for 
keeping domestic animals 

18 

Trial method to evaluate the efficacy of insecticidal 
products for the control of stable flies in premises for the 
rearing of domestic animals under practical conditions 

Vegephyl 

MS 1911, part 1 

Household insecticide products - 
evaluation method for biological 
efficacy - part 1: glass chamber 
method 

18 

This standard specifies a method for the evaluation of the 
biological efficacy of household insecticide products using 
the glass chamber method. This test method provides a 
satisfactory means to determine the relative effectiveness 
of common household insecticide products, namely 
mosquito electric vapourising liquid, mosquito vapourising 
mat and mosquito coils. The method is suitable for testing 
the above insecticide products against mosquitoes. 

MS 23 

Household insecticide products - 
Mosquito coil - Chemical, physical 
and biological efficacy 
requirements (Fifth revision) 

18 

This Malaysian Standard specifies the minimum chemical 
and physical requirements, and biological efficacy of 
mosquito coil products intended for household use against 
mosquitoes. 

JSM 

JSM 

   
 
 
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Reference 

Title 

PT 

Short test description 

449 

Reference 
source 

OPPTS 810.3400 

Mosquito, black fly, and biting 
midge (sand fly) treatments 

18 

Test of insecticides against flying insects: Mosquito, Black 
Fly and Biting Midge (Sand Fly) 

US EPA 

US CSMA Aerosol 
Guide, 7 th Edition, 
(1981), p. 129-134 

Test method for aerosol space 
sprays against flying insects 

18  Test of insecticides against flying insects: 

WHO/VBC/81.812 

Mosquito larvae resistance to 
insect development inhibitors 

18  Test kit and instruction sheet 

WHO/VBC/81.806 

WHO/VBC/81.807 

Instructions for determining the 
susceptibility or resistance of adult 
mosquitoes to organochlorine, 
organophosphate and carbamate 
insecticides 

Instructions for determining the 
susceptibility or resistance of 
mosquito larvae to insecticides 

18  Test kit and instruction sheet 

18  Test kit and instruction sheet 

WHO/VBC/81.811 

Blackfly larvae 

18  Test kit and instruction sheet 

WHO/VBC/81.813 

Houseflies, tsetse flies, stable 
flies, blowflies, etc. 

18  Test kit and instruction sheet 

WHO/CVB/81.5 

Test kit for bioassays on wall 
surfaces 

18  Test kit and instruction sheet 

WHO/CDS/CPC/MAL
/98.12 

Test procedures for insecticide 
resistance monitoring in malaria 
vectors, bio-efficacy and 
persistence of insecticides on 

18  Report of the WHO informal consultation 

CSMA 

WHO 

WHO 

WHO 

WHO 

WHO 

WHO 

WHO 

 
 
 
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Reference 

Title 

PT 

Short test description 

treated surfaces 

WHO/CDS/WHOPES
/GCDPP/2003.5 

Space spray application of 
insecticides for vector and public 
health pest control – a 
practitioner’s guide 

18 

Brief description of the main types of space spray 
equipment as well as the operational guidelines for space 
spray application of insecticides. 

WHO/CDS/WHOPES
/GCDPP/2005.13 

Guidelines for laboratory and field 
testing of mosquito larvicides 

18 

This document provides specific and standardized 
procedures and guidelines for testing larvicides, including 
bacterial larvicides and insect growth regulators against 
mosquitoes. 

WHO/CDS/NTD/WH
OPES/GCDPP/2006.
3 

Guidelines for testing mosquito 
adulticides for indoor residual 
spraying and treatment of 
mosquito nets 

18 

This document provides specific and standardized 
procedures and guidelines for testing mosquito adulticides 
for indoor residual spraying and for treatment of mosquito 
nets. 

WHO/HTM/ 
NTD/WHOPES/2009
.2 

Guidelines for efficacy testing of 
insecticides for indoor and outdoor 
ground-applied space spray 
applications 

18  The document provides guidance and stepwise procedures 
on laboratory studies, field testing and evaluation leading 
to the determination of efficacy, and application rates of 
insecticides for operational use in indoor and outdoor 
ground-applied space spray applications. With some 
modifications the guidelines can be used to determine 
efficacy against other flying vectors and pests.  

Reference 
source 

WHO 

WHO 

WHO 

WHO 

Table 48: Insecticides Against Textile and Stored Product Pests 

Reference 

Title 

CEB 135 BIS 

PT 

Short test description (if test method available or 
information  provided from elsewhere) 

18 

Method for the laboratory study of the efficacy of 
insecticide preparations intended for the treatment of 

Type of 
Reference 
Source 

Vegephyl 

   
 
 
 
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Reference 

Title 

PT 

Short test description (if test method available or 
information  provided from elsewhere) 

451 

Type of 
Reference 
Source 

CEB 213 

CEB 224 

PP1/201 

PP1/202 

PP1/203 

PP1/204 

NF G39-011 

18 

18 

18 
+ 
20 

18 

18 
+ 
20 

18 

18 

Fumigants to control insect and 
mite pests of stored plant 
products 

Space and structural treatments 
of store rooms 

Admixture of plant protection 
products to stored plant products 
to control insects and mites 

Laboratory testing of plant 
protection products against insect 
and mite pests of stored plant 
products 

Properties of textiles - Textiles 
and polymeric materials having 
anti-dustmite activity - 
Characterisation and 
measurement of anti-dustmite 

industrial processing storage premises and the marketing 
of products of animal or plant origin 

Method for studying the effectiveness of a fumigant for 
the disinsection of premises for the storage, processing 
and production of foodstuffs. 

Method for studying the efficacy of fumigants for the 
disinsection of stored foodstuffs. 

Vegephyl 

Vegephyl 

EPPO 

EPPO 

EPPO 

EPPO 

AFNOR 

This document describes a general method for 
determining the anti-mite activity of textiles and 
polymeric materials. The method is applicable to all 
textiles and polymeric materials with anti-mite activity, 
except those whose structure does not allow the contact 
defined under the test conditions. 

 
 
 
 
 
 
 
 
 
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Reference 

Title 

PT 

Short test description (if test method available or 
information  provided from elsewhere) 

Type of 
Reference 
Source 

NF ISO 3998 

Textiles- Determination of 
resistance to certain insect pests 

18 

This standard specifies a method for the determination of 
the resistance of textiles to the larvae of certain insects.  

AFNOR 

ISO 3998 

Textiles - Determination of 
resistance to certain insect pests 

18 

Applicable to all textiles containing animal fibres in any 
proportion. Comparing the resistant material against a 
non-resistant material. 

ISO 

Table 49: Repellents & Attractants 

Reference 

Title 

PT 

Short test description 

Reference 
source 

Ctgb 

Evaluation manual for the 
authorization of biopesticides 
according to regulation (EC) No. 
1107/2009, microorganisms, 
botanicals, semiochemicals. 

19 

This document describes in more detail the data 
requirements and risk assessment for biopesticides. 

Ctgb 

CVMP/411/2001 

Specific efficacy requirements for 
ectoparasiticides in sheep 

19 

CVMP/625/2003 

Specific efficacy requirements for 
ectoparasiticides in cattle 

19 

This document provides guidance on the study of the 
efficacy of products against the principal parasites found 
in sheep. It can be applied also to products against less 
common (regional) ectoparasites, providing that any 
adjustments to the methods are justified. 

This document provides guidance on how to study the 
efficacy of products in cattle against all arthropod species 
that need animal involvement for completing their life-
cycle. 

EMEA/CVMP/EWP/0
05/2000-Rev.3 

Guideline for the testing and 
evaluation of the efficacy of 
antiparasitic substances for the 

19 

This guideline provides specific guidance with respect to 
the testing and evaluation of efficacy of veterinary 
antiparasitic products that are intended for the treatment 

EMA 

EMA 

EMA 

   
 
 
 
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Reference 

Title 

PT 

Short test description 

453 

Reference 
source 

treatment and prevention of tick 
and flea infestation in dogs and 
cats 

Wood preservatives - 
Determination of the protective 
effectiveness of a preservative 
treatment against blue stain in 
wood in service - Laboratory 
method 

and prevention of tick and flea infestations in dogs and 
cats, and includes information for the testing of veterinary 
systemically and locally acting antiparasitic products and 
products containing substances with insect growth 
regulating properties (IGRs), either as mono-preparations 
or in combination with an adulticide. I 

This European Standard specifies a method which is only 
suitable for testing preparations and systems which are 
intended to prevent the occurrence of blue stain fungi in 
wood in service. This European Standard lays down a 
method for determining the effectiveness of a preparation 
applied by e.g. brushing, spraying, spraying tunnel, 
dipping or vacuum and pressure treatments resulting in 
an equivalent retention of product in preventing the 
development of blue stain fungi in wood in service. 

Protective clothing against rain - 
Test method for ready-made 
garments - Impact from above 
with high energy droplets 

Textiles - Domestic washing and 
drying procedures for textile 
testing 

19 

19 

This European Standard specifies a test method for 
determining the rain tightness of clothing for protection 
against rain, using a static manikin exposed to artificial 
rain. It is applicable to the testing of jackets, trousers, 
coats and one or two piece suits. 

This standard specifies domestic washing and drying 
procedures for textile testing. The procedures are 
applicable to textile fabrics, garments or other textile 
articles which are subjected to appropriate combinations 
of domestic washing and drying procedures.  

CEN 

CEN 

ISO 

EN 152 

EN 14360 

ISO 6330 

ENV/JM/MONO(200
0)7 

Guidance Document on the 
Recognition, Assessment and Use 
of Clinical Signs as Humane 
Endpoints for Experimental 

19 

The purpose of this guidance is to apply the principles of 
the Three Rs (Replacement, Reduction, Refinement) to 
the use of animals in regulatory toxicity tests 

OECD 

 
 
 
 
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Reference 

Title 

PT 

Short test description 

ENV/JM/MONO(200
1)12 

Animals used in Safety Evaluation 

Guidance for Registration 
Requirements for Pheromones and 
Other Semiochemicals Used for 
Arthropod Pest Control 

ISBN 978-1-
891127-75-5 

Atlas of Stored-Product Insects 
and Mites 

19 

19 

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OECD 

AACC 
International 

OPPTS 810.3300  

Treatments to control pests of 
humans and pets 

This guideline is concerned with efficacy testing of 
invertebrate control pesticides used on humans and pets. 

US EPA 

PP1/152 

Design and analysis of efficacy 
evaluation trials 

PP1/181 

Conduct and reporting of efficacy 
evaluation trials, including good 
experimental practice 

19 

19 

This standard is intended to provide general background 
information on the design and analysis of efﬁcacy 
evaluation trials. 

This standard provides guidance on how to organize trials, 
and how to plan, conduct and assess them, then record 
and interpret them, so as to obtain comparable and 
reliable results. 

PP1/264(2) 

Principles of efficacy evaluation for 
mating disruption pheromones 

19 

This standard describes the general principles of trial design 
for the efﬁcacy evaluation of mating disruption techniques 
based on pheromones. These techniques are based on female 
sex pheromones, but others could also be used, e.g. 

aggregation pheromones which attract both sexes for 

mating or, in rare cases, where males produce sex 
pheromones. 

EPPO 

EPPO 

EPPO 

PP1/296 

Principles of efficacy evaluation for  19  The objective of this document is to provide a framework 

EPPO 

   
 
 
 
 
 
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for the minimum efﬁcacy data requirements needed to 
demonstrate that a low-risk plant protection product is 
sufﬁciently effective (and crop safe) for authorization. 

The document contains default values for the room in 
which the exposure takes place and for the person that is 
exposed. In addition, it presents information on the 
ventilation in houses, inhalation rates and data on activity 
patterns.  

low-risk plant protection products 

RIVM report 
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consumer exposure - Updated 
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SANCO/11470/201
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SANTE/12815/2014 
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Alcaine-Colet A. et 
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Arnault I. et al. 

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adapted four arms olfactometer 

19 

19 

19 

19 

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attractiveness of plant volatiles to 
adult Helicoverpa zea 

Addition of Alarm Pheromone 
Components Improves the 
Effectiveness of Desiccant Dusts 
Against Cimex lectularius 

19 

Benoit J.B. et al. 

Blume R.R. et al. 

Tests of aerosols of deet for 
protection of livestock from biting 
flies 

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Büchel, K., Kleier 
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highly standardized laboratory 
bioassay for repellent against cat 
fleas 

Büchel K. et al. 

Carroll J.F. et al. 

Repellent efficacy of DEET, 
Icaridin, and EBAAP against 
Ixodes ricinus and Ixodes 
scapularis nymphs (Acari, 
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Comparative Activity of DEET and 
AI3-37220 Repellents Against the 
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Prolonged Efficacy of IR3535 
Repellents Against Mosquitoes and 
Blacklegged Ticks in North 
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Rapid risk assessment: 
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Spain and France 

19 

Carroll S.P. 

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Dautel H et al. 
International 
Journal of Medical 
Microbiology, Vol 
293, Supplement 
37, April 2004, 
pages 182-188 

A novel test system for detection 
of tick repellents  

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The so-called Moving Object Bioassay is described, a tool 
for testing the strength of potential tick repellents 
quantitatively. Endpoint measured is the attachment rate 
of Ixodes ticks. 

International 
Journal of 
Medical 
Microbiology 

Dweck H. et al. 

Olfactory Proxy Detection of 
Dietary Antioxidants in Drosophila 

19 

Fradin M., Day J. 

Comparative efficacy of insect 
repellents against mosquito bites 

19 

Geier M., Boeckh J. 

A new Y-tube olfactometer for 
mosquitoes to measure the 
attractiveness of host odours 

19 

Human subjects: Arm in cage studies (15 volunteers, 10 
mosquitoes (Aedes aegypti) in each cage. Endpoint: 
elapsed time to first bite. Category of protection A-H 
(significantly different mean complete protection time; 
ANOVA & Tukey's). No need to recalculate the results to 
"real condition" (simulate real condition) 

Current 
Biology 
25(4): p. 
455-466 

The New 
England 
Journal of 
Medicine 
347(1): p. 
13-18 

Entomologia 
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Repellents  

Herholz C. et al. 

Efficacy of the repellent N,N-
diethyl-3-methyl-benzamide 
(DEET) against tabanid flies on 
horses evaluated in a field test in 
Switzerland 

19 

19 

Hill J.A., Robinson 
P.B., McVey D.L., 
Akers W.A. et al. 

Evaluation of mosquito repellents 
on the hairless dog 

19 

Ja W.W. et al. 

Prandiology of Drosophila and the 
CAFE assay 

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160, CRC 
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Parasitology 
221: p. 64-
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Mosquito 
news 39(2) 

Proceedings 
of the 
National 
Academy of 
Science, 
104(20): p. 
8253-8256 

Japin, M. and 
Haanen, G. A. Y. 

Culicoides species in the 
Netherlands: a comparison 
between tent traps and an 
Onderstepoort black light trap and 
the effect of an insect blanket on 

19 

The aims of the present study were to determine which 
species in which numbers of Culicoides (that potentially 
serve as vectors for AHSV) are attracted to horses in the 
Netherlands and to compare these results with the 
Culicoides species and numbers caught in the 

Faculty of 
Veterinary 
Medicine 
Theses 
(2013) 

   
 
 
 
 
 
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the biting rat 

Onderstepoort black light trap during the same period. 
The second aim was to evaluate the use of an insect 
blanket on the biting rate of Culicoides species. 

Kline D.L., Mann 
M.O. 

Evaluation of butanone, carbon 
dioxide, and 1-octen-3-ol as 
attractants for mosquitoes 
associated with North Central 
Florida bay and cypress swamps 

19 

Knaden M. et al 

Spatial Representation of Odorant 
Valence in an Insect Brain. 

19 

Krüger, A., S. 
Knobelspieß, and E. 
Schmolz 

Development and evaluation of 
testing methods for ant repellents 

19 

This document describes two test systems for efficacy 
evaluation of ant repellents with the substances DEET 
50%, Margosa extract 100%, baking powder: sodium 
hydrogen carbonate and sea sand. They are designed as 
choice tests and allow testing of solid and liquid 
substances. 

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American 
Mosquito 
Control 
Association 
14(3): p. 
289-297 

Cell 1(4): p. 
392-399 

9th 
International 
Conference 
on Urban 
Pests, 
Birmingham. 
2017 

Marchiondo A.A., et 
al. 

World Association for the 
Advancement of Veterinary 
Parasitology (W.A.A.V.P.) second 
edition: guidelines for evaluating 
the efficacy of parasiticides for the 
treatment, prevention and control 
of flea and tick infestations on 

19 

These guidelines are intended to assist the planning and 
conduct of laboratory and clinical studies to assess the 
efficacy of ectoparasiticides applied to dogs or cats for the 
purpose of treating, preventing and controlling flea and 
tick infestations. 

Veterinary 
Parasitology, 
194(1): p. 
84-97 

 
 
 
 
 
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dogs and cats 

Mottet et al. 

Effectiveness of stable fly 
protectants on adult horses 

19 

Moreno-Gómez M. 
et al. 

From the field to the laboratory 
quantifying outdoor mosquito 
landing rate to better evaluate 
topical repellents 

19 

Moreno-Gómez M. 
et al. 

Two new alternatives to the 
conventional arm-in-cage test for 
assessing topical repellents 

19 

Mulatier M. et al. 

DEET Efficacy Increases With Age 
in the Vector Mosquitoes 
Anopheles gambiae s.s. and Aedes 
albopictus (Diptera: Culicidae) 

19 

This study aimed to estimate the landing rate outdoors, in 
an area of Europe highly infested with the Asian tiger 
mosquito, Aedes albopictus, and to determine how to 
replicate this rate in the laboratory. This study provides 
useful reference values that can be employed to design 
new evaluation standards for topical repellents that avoid 
field conditions given that the latter exposes study 
participants to health risks. 

Two alternative laboratory methods that use mosquito 
landing rates more representative of those in the field 
were assessed. These methods showed to be potential 
alternatives to the current AIC method, as well as being a 
better proxy for recreated field mosquito landing rates., 
reduced variability among study participants, and 
achieved reproducible protection times across 
laboratories. 

Reference 
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Journal of 
Equine 
Veterinary 
Science 69: 
p. 11–15 

Journal of 
Medical 
Entomology; 
tjaa298 

Journal of 
Medical 
entomology; 
tjab050 

Journal of 
Medical 
Entomology 
55(6); p. 
1542-1548 

   
 
 
 
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Obermayr U. et al. 

A novel test cage with an air 
ventilation system as an 
alternative to conventional cages 
for the efficacy testing of 
mosquito repellents 

19 

Okumu F. et al. 

Using nylon strips to dispense 
mosquito attractants for sampling 
the malaria vector Anopheles 
gambiae s.s. 

19 

Plarre R. et al. 

Effects of oil of cloves and 
citronellol, two commercially 
available repellents, against the 
webbing clothes moth Tineola 
bisselliella Hum. (Lepidoptera: 
Tineidae) 

19 

Smith C.N. et al. 

Factors Affecting the Protection 
Period of Mosquito Repellents 

19 

Stensmyr M. C. et 
al. 

A Conserved Dedicated Olfactory 
Circuit for Detecting Harmful 
Microbes in Drosophila 

19 

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Entomology 
47(6): p. 
1116-1122 

Journal of 
Medical 
Entomology 
47(2): p. 
274-282 

Journal of 
Pest Science 
70(3): p. 45. 

U.S. 
Department 
of Agriculture 

Cell 151(6): 
p. 1345-1357 

 
 
 
 
 
 
 
 
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Hummel, E., 
Kleeberg, H. 1997. 
in: Practice 
orientated results 
on use and 
production of 
Neem-Ingredients 
and Pheromones V. 
Proceedings of the 
5th workshop, 
Wetzlar, Germany, 
January 22-25, 
1996 

Effect of the neem extract 
formulation neemazal-t/s on the 
green pea aphid acyrthosiphon 
pisum in the laboratory (1995), 
in: Practice orientated results on 
use and production of Neem-
Ingredients and Pheromones V 

19 

Vander Pan A. et al. 

A Novel Simulated-Use Test for 
Determining the Efficacy of 
Insecticides Against Bed Bugs 
(Hemiptera: Cimicidae) 

19 

The objective of this study was to develop a simulated-
use test system for efficacy testing of insecticides with 
residual properties against bed bugs, imitating a typical 
insecticide barrier treatment under practical conditions. 

Vythilingam I. et al. 

Evaluation of carbon dioxide and 
1-octen-3-ol as mosquito 
attractants 

19 

Wade S.E., Georgi 
J.R. 

Survival and reproduction of 
artificially fed cat fleas, 
Ctenocephalides felis Bouché 

19 

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Trifolio-M 
GmbH 

Journal of 
Economic 
Entomology 
112(5), p. 
2345-2353 

The 
Southeast 
Asian Journal 
of Tropical 
Medicine and 
Public Health 
23(2): p. 
328-331 

Journal of 
Medical 
Entomology, 

   
 
 
 
 
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(Siphonaptera: Pulicidae) 

Wang C. et al. 

Repellency of selected chemicals 
against the bed bug (Hemiptera: 
Cimicidae) 

SANS 5695 

Pesticides – Biological properties - 
Efficacy of mosquito repellents 

OPPTS 810.3700  

Insect repellents to be applied to 
human skin 

19 

19 

19 

WHO 

Guidelines for efficacy testing of 
spatial repellents 

19 

Specifies 3 methods for the biological evaluation of the 
efficacy of mosquito repellents. 

The document provides guidance and describes steps for 
laboratory testing and for semi-field and field evaluations 
of spatial repellent products (technical materials and 
formulated products) designed to provide protection in a 
specific space (indoor and/or outdoor) against 
mosquitoes. 

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190, 

Journal of 
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106(6) 2013: 
p. 2522-2529 

SABS 

US EPA 

WHO 

WHO/HTM/NTD/WH
OPES/2009.4 

Guidelines for efficacy testing of 
mosquito repellents for human 
skin 

19  The purpose of these guidelines is to provide specific and 

WHO 

standardized procedures and criteria for efficacy testing 
and evaluation of mosquito repellents for human skin. 
Their aim is to harmonize the testing procedures carried 
out in different laboratories and institutions in order to 
generate comparable data for registering and labelling 
such products by the national regulatory authorities. 

MS 1497 

Household insecticide products - 
personal mosquito repellent - 
evaluation method for biological 

19 

Methods of biological evaluation of the efficacy of 
repellent - bioassay method for mosquito repellent on 
human skin 

Malaysian 
Institute of 
Chemistry 

 
 
 
 
 
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efficacy 

7AE17a 

Demonstration of Efficacy of 
Ectoparasiticides 

19 

This document provides general requirements for the 
assessment of efficacy of an ectoparasiticide preparation, 
containing novel or established active ingredients. 

EMA 

   
 
 
 
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Appendix 19. Efficacy  guideline  with  Cockroach;  field 
trial 

This  guidance  describes  an  example  of  a  field  trial  to  determine  efficacy  of  a  product 
against the German cockroach (Blattella germanica). 

Global design 

In  a  pre-test  it  is  established  whether  the  population  of  cockroaches  in  an  object  is 
large enough for a field trial. An indication of the population size is obtained in the pre-
test by using a spray with expelling action or by setting glue traps.  

If  the  population  size  is  large  enough,  a  pest  control  operation  is  performed.  The 
efficacy  of  the  product  is  determined  by  measuring  the  population  size  again  8  weeks 
later and comparing it to the initial value. 

During  these  8  weeks  the  effect  of  the  control  operation  should  be  checked  at  least  4 
times  at  regular  intervals  (possibly  using  glue  traps).  The  investigator  himself  should 
perform these checks during the trial. 

Requirements  for  the  practical  use  situation  in  order  to  be  suitable  as  test 
object.  

The field trial is performed in three separate objects. 

Recommendations for the practical use situation to produce a good field trial for control 
of the German cockroach are as follows: 

1.  History  of  insecticide  use  should  be  described  with  as  much  detail  as  possible 
(which  product,  active  ingredient,  when  …).  Object  with  recent  insecticide  use 
should not be included in the test. 

2.  The test object  should  preferably and where possible be hermetically  sealed  off 
from  the  surrounding  buildings.  If  there  are  adjacent  buildings,  all  cracks  and 
crevices  on  the  outside  of  the  test  object  should  be  treated  with  an  authorised 
biocidal product with residual action. 

3.  The test object should preferably contain at least a kitchen or kitchen unit, with 

one or more refrigerators or freezers. 

4.  Cockroaches should be present in the test object, both in the kitchen or kitchen 

unit as elsewhere. 

5.  In the preceding 8 weeks no other chemical control of cockroaches should have 

taken place in the test object. 

Field trial 

The pre-test 

Aim: To determine whether the population is large enough for a field trial. 

Execution: Within 1 week before the control operation. 
The pre-test can be conducted in two different ways. 

1.  By using a spray liquid with an expelling action (e.g. pyrethrins): 

Spray under the refrigerator and one other place in the kitchen where there are 
probably many cockroaches. 
Spray for 3 seconds and count the cockroaches that emerge during 1 minute. 

 
 
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2.  By using glue traps 

Place glue traps at places where many cockroaches are expected.  
Number per unit area: 5 glue traps per 100 m2 
Describe  clearly  where  the  glue  traps  are  placed,  and  record  the  number  of 
trapped  cockroaches  after  an  appropriate  period,  usually  either  overnight,  or 
after  up  to  3  days  (e.g.  weekend),  depending  upon  the  scale  of  the  infestation 
(shorter trap periods for heavier infestations to avoid traps becoming saturated 
and  failing  to  catch  cockroaches  later  during  the  monitoring  period;  longer 
periods  when  infestation  level  is  low  and  few  cockroaches  are  trapped  each 
night). 

Criteria for a suitable test object 

•  When  a  trap  is  placed  for  48  hours  in  the  kitchen  or  in  the  kitchen  unit  behind 
the refrigerator, it should contain at least 10 adult cockroaches at the end of this 
time, as well as several nymphs. 

•  Several cockroaches should be caught on at least one glue trap, which is placed 
at another place in the kitchen or kitchen unit and on one trap, which is placed 
outside the kitchen or kitchen unit, within 48 hours. 

Or 

•  When using a spray with expelling action, at least 5-10 cockroaches per sprayed 

site should be counted. 

The test 

Duration of the control period until measurement of efficacy is about 8 weeks. 

The pest control is performed according to the directions for use of the product. 

During these 8 weeks the investigator will check the progress of the  control at least  4 
times. 

Directions for use of an insecticide in the form of a spray liquid: 

It should be clear how much product is used, on average 1 L/20 m2 is sprayed; 

• 
•  Treatment of cracks and crevices should be done where necessary; 
• 

If stated on the label, a second treatment can be performed. 

Directions for use of an insecticide in the form of a powder: 

• 

It should be clear how much product is used. 

Directions for use of an insecticide in the form of bait: 

•  Number of baits placed per unit area should be according to directions for use; 
•  Precise descriptions of where the baits are placed should be given; 
•  The baits that are placed remain in situ for 8 weeks continuously, unless stated 

differently on the label.  

Required results 

At  least  4  times  during  the  test  and  at  the  end  of  the  test  (about  8  weeks  after  the 
start), an estimate of the population size is obtained in the same manner as during the 
pre-test. The difference in population size before and 8 weeks after the control operation 
provides the degree of efficacy of the product. 

 
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Appendix 20. Current Antifouling Coatings 

The current major types of antifouling coatings are outlined below, together with a brief 
description  of their properties. This list is not  exhaustive, and product applications may 
not fall within these categories. Applicants may submit novel coating  types not  covered 
by this list. 

Table 50: Current Antifouling Coatings 

Coating 
Type  

Soluble 
matrix  

Insoluble 
matrix  

Self 
polishing  

Description, mode of action and properties  

In  coatings  of  this  type  the  active  substance(s)  has  (have)  been  physically 
mixed  (‘freely  associated’)  into  a  resin  matrix.  Upon  exposure  to  seawater  the 
slightly acidic matrix  slowly dissolves  releasing the active substance(s) into the 
water.  (Seawater  is  slightly  alkaline  (pH  8)  and  the  acidic  matrix  dissolves). 
Continuous dissolution of the coating surface will occur resulting in fresh actives 
being released until eventually the film is  exhausted. Soluble matrix  antifouling 
products typically show a biocide release rate curve which decays exponentially. 

The soluble matrix coatings have reduced mechanical properties that limit their 
film thickness. The paint film thickness of these coatings depletes over time in a 
fairly imprecise manner and the film does not show smoothing characteristics on 
ships  in  service.  Such  coatings  are  normally  specified  for  lifetimes  of  typically 
12-36 months. 

This type of coating contains a mixture of resins that together form an insoluble 
binder  phase.  One  or  more  active  substances  are  physically  mixed  into  this 
matrix.  As  seawater  enters  the  paint  film,  the  biocides  are  released  by 
dissolution and diffusion from within the insoluble matrix. After active substance 
have  been  released  from  the  film,  the  binder  remains  intact  and  an  empty 
‘honeycomb’  structure  (the  leached  layer)  remains  at  the  paint  surface.  This 
type  of  coating  has  a  high  initial  release  rate,  which  decreases  exponentially 
with time as the active substance(s) have further distance to travel through the 
paint  film.  The  rate  of  diffusion  of  biocide  from  within  the  film  then  becomes  a 
limiting  factor  in  maintaining  an  effective  biocide  release  rate  and  hence 
preventing fouling. 

Insoluble  matrix  antifouling  coatings  do  not  show  film-depletion  or  polishing  as 
the  resin  is  insoluble.  The  biocide  release  process  continues  until  exhaustion  of 
the coating. The higher mechanical strength obtained with these coatings allows 
for  applications  of  thicker  systems  and  coating  lifetimes  of  typically  12-  36 
months are attainable. 

This  group  is  currently  the  most  common  and  covers  a  range  of  different 
technologies 
through  a  gradual 
the  active 
depletion/ablation of the paint film throughout the lifetime of the coating. 

that  deliver 

substance 

These  coatings  use  binder  systems  which  control  polishing  behaviour  by 
different  mechanisms.  A  broad  range  of  binder  technologies  are  found  in  this 
group  and  these  have  replaced  TBT  copolymer  based  paints  which  have  been 
withdrawn from use. Binder systems range from those based on the dissolution 
of metal carboxylates and polymers relying on ion-exchange to polymers relying 
on hydrolysis to control the rate of polishing. 

Modification  of  the  binder  systems  and  pigment  phases  of  products  within  this 

 
 
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Description, mode of action and properties  

group  can  be  used  to  tailor  the  products  towards  different  end  uses.  The 
requirements for protection of a fast moving and very active vessel can be very 
different from that of a slow moving less active one. Such modifications can also 
be used to tailor performance to accommodate the potential intensity of fouling. 

The different binder technologies can be used alone or in combination and result 
in  products  with  varying  levels  of  antifouling  protection.  Other  binder 
components may also be added in order to modify the overall properties of the 
paint  film.  Typical  dry-docking  intervals  for  vessels  coated  with  self  polishing 
antifouling paints range from 24 to 60 months, however these systems may also 
be specified for lifetimes beyond this period. 

 
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469 

Appendix 21. Published paper (CEPE Antifouling Working 
Group) 

NOTE to the reader: 

In the following CEPE methodology there are several issues that contradict with the 
requirements  in  the  guidance  document  (e.g.  number  of  trial  panels,  period  of 
testing).  The  CEPE  methodology  can  be  used  as  long  as  the  agreements  of  the 
guidance are respected. 

TMI2013-PT21_efficacy_workshop-CEPE  Efficacy  Methodology  for  BPR  - 
Revised 19 June 2012.doc 

The European Council of producers and importers of paints, printing inks and artists’ 
colours - CEPE 
Guidance developed by the CEPE Antifouling Working Group 

Efficacy evaluation of antifouling products  

Conduct and reporting of static raft tests for antifouling efficacy 

Specific scope 

This document provides a baseline methodology for evaluating and reporting the efficacy 
of  antifouling  coatings.  Efficacy  is  assessed  by  static  raft  testing  relative  to  a  negative 
control and, if used, a positive control coating. Efficacy may be indicative of, but has no 
direct  one-to-one  relationship  with  the  actual  performance  of  a  product  under  real  life 
conditions. 

Document version 

First approved in 2011-04. 

Revised in 2012-06 

1. 

Scope 

Overview:  The  purpose  of  this  document  is  to  provide  a  methodology  for  determining 
efficacy  of  antifouling  coatings  by  panel  testing  on  static  floating  rafts.  The  document 
provides  guidance  on  how  to  conduct,  assess,  record,  and  report  results  from  efficacy 
evaluations. 

Efficacy  is  evaluated  relative  to  a  suitable  inert,  negative  control.  A  positive  control  of 
proven antifouling performance may also be included. This static exposure methodology 
for  natural  environments  is  not  suitable  for  establishing  absolute  performance 
characteristics of antifouling coatings in service. 

Objective: This methodology may be used by industry to obtain efficacy data during the 
development of new antifouling coatings. This methodology may also be used to provide 
national registration authorities with the information required to support the label claim 
of antifouling products. Efficacy is demonstrated when the extent of fouling is visibly less 
than on a blank panel. 

The methodology is especially useful for: 

• 

• 

the persons responsible for writing the protocols for antifouling efficacy trials 

the  persons  responsible  for  conducting  trials  including  the  evaluation  and 
recording of results 

 
 
 
 
 
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• 

• 

the  persons  responsible  for  assembling  and  submitting  dossiers  for  the 
registration of antifouling paints 

the  national  authorities  which  are  responsible  for  the  assessment  of 
registration dossiers. 

Reproducibility  and  accuracy:  In  static  raft  testing  the  fouling  intensity  will  vary 
significantly  between  different  geographical  locations,  between  positions  on  the  same 
rafts,  and  from  season  to  season.  More  importantly,  fouling  will  vary  from  one  year  to 
the  next  even  for  identical  panels  where  exposure  starts  around  the  same  date  in 
different  years.  This  variability  in  fouling  intensity,  and  thus  the  test  results,  is  due  to 
weather  conditions,  availability  of  nutrients,  and  other  uncontrollable  factors  that  may 
affect  the  type  and  extent  of  fouling  and  its  rate  of  settlement  and  growth.  Therefore, 
the  absolute  amount  of  fouling  present  on  the  test  coating  and  controls  may  not  be 
reproducible at the same site from year to year. 

Interpretation  of  results:  The  results  obtained  by  this  methodology  demonstrate  the 
ability  of  antifouling  coatings  to  prevent  settlement  of  fouling  organisms  under  static 
conditions  relative  to  a  suitable  negative  control  and,  if  used,  a  positive  control  tested 
simultaneously  at  the  same  site.  An  evaluation  of  the  relative  antifouling  effect  of  an 
antifouling coating compared to the negative control and, if used, the positive control is 
used  as  a  tool  to  indicate  the  potential  of  a  tested  coating  to  protect  underwater 
structures. The results can be used to support appropriate label claims of the antifouling 
coating tested and to screen for new candidate products. 

Efficacy  testing  on  raft  panels  represents  a  worst  case  scenario  compared  to  real  life 
conditions.  The  main  reason  is  that  the  exposure  is  static  with  limited  opportunity  for 
organisms to be removed by hydrodynamic forces. Ships' and boats' movement through 
water  also  aid  the  release  of  active  ingredients  from  their  antifouling.  Furthermore, 
fouling  intensity  is  generally  recognised  as  being  greater  near  the  coast  relative  to  the 
open seas. 

2. 

Definitions 

Antifouling  coating:  A  material  which,  when  applied  as  a  surface  coating,  is  used  to 
control  the  settlement  and/or  growth  of  fouling  organisms  on  submerged  surfaces 
including  ships,  boats,  aquaculture  equipment,  offshore  oil  installations,  and  other  man 
made structures. 

Negative  control:  An  inert  reference  surface  that  does  not  control  fouling,  e.g.  an  anti-
corrosive coating. 

Positive  control:  A  reference  surface  coated  with  an  antifouling  coating  of  appropriate 
efficacy relevant to the intended end use of the test coating. 

Fouling season: The months of the year during which significant settlement and growth 
of fouling organisms typically occur on a negative control at the test site. 

3. 

Apparatus 

The  following  equipment will be  required to undertake efficacy testing according to this 
methodology. 

Panels:  Panels  are  typically  made  of  plastic  (e.g.  PVC),  reinforced  polyester,  steel, 
aluminium, marine grade plywood, or other material suitable for extended immersion in 
natural  waters.  (Metal  panels  must  be  adequately  protected  with  an  anticorrosive  paint 
system.) 

Panels  should  be  designed  to  allow  them  to  be  securely  fixed  to  the  test  raft,  for 
example  via  a  suitable  panel  rack.  Where  the  design  requires  fixing  holes  through 
panels,  these  holes  should  be  drilled  prior  to  the  application  of  the  coating  to  prevent 
damage. 

 
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471 

The panels may be designed to allow one or more coatings and/or controls to be tested 
on  each  individual  panel.  The  total  immersed  area  of  each  coating  or  control  should  be 
no less than 100 cm2. 

Raft: A free floating platform which has been designed to allow test panels to be affixed 
and  immersed  at  a  constant  depth  in  natural  waters.  The  design  of  the  raft  should 
enable panels to be readily removed for inspection. 

The minimum depth of water below the raft at low tide should generally be 2.5 m. 

The  floating  raft  should  be  of  sufficiently  rigid  construction  to  withstand  prolonged 
exposure to weather and wave action and prevent excessive flexing or movement of test 
panels. It should be designed to ensure the occupational safety of users. 

The raft should be designed to ensure that all test coatings and controls of the same test 
series are exposed to similar levels of sunlight and water flow to minimise variation. To 
increase  the  testing  capacity,  panels  may  be  affixed  to  the  raft  in  rows  at  the  same 
depth.  Where  relevant  the  spacing  between  parallel  rows  at  the  same  depth  should 
generally be at least 20 cm to allow sufficient water circulation and illumination. 

Generally,  the  raft  design  should  ensure  that  panels  are  fully  and  permanently 
immersed. Panels should normally be exposed vertically and at a fixed depth from 0-3 m 
below  the  water  surface.  The  lower  edge  of  the  panel  should  always  be  at  least  0.5  m 
above the sea bed. 

 The  raft may also be designed to allow  coatings that are intended for use in darker or 
lighter  areas  to  be  tested  under  relevant  conditions  where  the  coating  receives  less  or 
more  sunlight. In such cases panels may be mounted on the raft facing partly down  or 
up. Shade may also be provided by covering parts of the raft. 

4. 

Safety 

This  test  methodology  does  not  address  possible  safety,  health  and  environmental 
concerns associated with its use. All operations should be performed in accordance with 
all relevant local and national regulations. 

Personal  protection:  Antifouling  coatings  may  contain  hazardous  materials  that  could 
cause  skin  and  eye  irritation  on  contact  and  adverse  physiological  effects  if  inhaled. 
Thus, application and drying should take place in a well ventilated area  and appropriate 
personal  protective  equipment  should  be  worn  during  application.  Product  safety  data 
sheets should be consulted when available. 

Environmental  protection:  Unused  paint  and  other  contaminated  material  as  well  as 
panels after exposure should be disposed of as hazardous waste. 

5. 

Procedure 

All  controls  and  test  antifouling  coatings  should  be  tested  under  equivalent  conditions. 
The  exposure  (immersion)  of  controls  and  test  antifouling  should  start  simultaneously 
(around  the  same  date)  and  the  exposure  should  be  at  the  same  location  at  the  same 
depth and orientation. 

Panel  preparation:  The  test  coating  and  positive  control  should  be  applied  to  panels 
according  to  the  manufacturer's  guidelines  to  ensure  adhesion  during  the  period  of  the 
study.  Appropriate  drying  and  recoating  intervals  and  temperature  and  ventilation 
requirements for application of the coatings should be followed. 

An  appropriate  means  of  application  should  be  used.  Typical  methods  include  spray, 
roller,  brush,  or  specialised  application  equipment  like  a  bar  type  applicator.  Sufficient 
film  thickness,  taking  the  expected  polishing  and  leaching  rate  characteristics  of  the 
product  into  account,  should  be  applied  to  last  for  the  planned  duration  of  the  test. 
Unless both sides of a panel are  used as test  substrates, the back of  the panel may be 

 
 
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coated with an antifouling of proven efficacy to prevent fouling on the back. Edges may 
be painted with the coating under test or with a different coating of proven efficacy. All 
panels should be marked indelibly with a suitable reference code to aid identification.   

Replicates: In cases where the purpose of the test is simply to demonstrate the efficacy 
of a test coating relative to a negative control, the use of single panels may provide data 
of  sufficient  quality.  When  replication  is  used,  the  number  of  replicates  should  be 
appropriate for the specific purpose of the test and should have the same orientation as 
the test panels and controls. Read-across to efficacy data from other test panels in a test 
series  of  similar  formulations  with  the  same  content  of  active  ingredients  may  also  be 
used when justified and reasonable to support the results obtained for the test coating. 

Exposure time: To verify efficacy, the minimum immersion time for testing is six months. 
In  locations  where  the  fouling  season  is  shorter  than  six  months  this  period  may  be 
reduced.  The  efficacy  test  should  cover  at  least  one  continuous  and  complete  fouling 
season  where  appropriate.  Since  raft  panel  exposure  is  static,  fouling  intensity  is  high, 
and the tests may be regarded as an accelerated test for products for vessels. 

6. 

Evaluation 

Frequency:  Antifouling  coatings  under  test  and  controls  should  be  regularly  inspected 
and  evaluated  for  surface  fouling,  typically  about  every  two  months  during  the  fouling 
season. Evaluations are not necessary during periods where there is minimal settlement 
and  growth  of  fouling  organisms  (e.g.  in  cold  and  temperate  regions  where  winter 
conditions  do  not  support  fouling  settlement).  Generally,  the  panels  will  be  removed 
from the water for evaluation and, except at the end of the test period, returned to the 
water immediately after evaluation. 

Rinsing:  Optionally,  panels  may  be  rinsed  gently  with  water  from  the  site  in  order  to 
reduce  the  influence  of  non-sessile  organisms  (that  would  be  removed  by  low  shear 
forces).  Rinsing  may  also  be  carried  out  to  remove  possible  sedimentary  material  (clay 
or  silt).  If  utilised,  rinsing  must  be  performed  on  all  panels  equally  and  at  each 
inspection. The method chosen, or if panels are not rinsed, must be specified in the final 
report. 

Evaluation procedure: The type and severity of fouling that is present on the test coating 
and  controls  shall  be  assessed  at  each  inspection.  Evaluation  may  be  made  by  visual 
assessment  on  site  or  any  other  appropriate  method  e.g.  image  analysis.  The  three 
major  types  of  fouling  observed  on  the  test  coating  or  controls;  Slime,  algae,  and 
animals,  should  be  separately  assessed  since  the  same  percentage  of  coverage  may 
have very different economical penalties during actual in-service use (e.g. effect on the 
friction of a vessel through water). Also  fouling organisms that are known not to attach 
on  moving  vessels,  but  may  be  frequent  on  static  surfaces,  should  be  assessed 
separately (e.g. amphipods). 

Further  classification  of  the  fouling  organisms  present  may,  in  addition  to  slime 
(biological film of microfouling including bacteria, diatoms, micro-algae, and extracellular 
biopolymers), generally be restricted to main categories such as green, red, and brown 
macro-algae, bryozoa, hydrozoa, barnacles, tube worms, ascidians, and mussels. A more 
detailed  determination  is  generally  not  necessary  since  products  shall  prevent 
attachment of fouling irrespective of species (or other taxonomic ranking). 

As  the  assessment  is  based  on  a  visual  inspection,  it  is  advised  that  this  is  done  by  a 
trained operator. This will help to improve consistency and data quality.   

Assessment  for  the  severity  of  fouling  for  each  type  of  organism  should  be  semi-
quantitative, for example using a scale from 0-4, where 0 indicates the absence, and 4 
indicates  complete  coverage  of  the  class  of  organism  in  question.  Optionally  an 
estimation of the percentage coverage can be used. 

 
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473 

The  assessment  of  the  coverage  of  algae  and  other  soft  fouling  (e.g.  arborescent 
bryozoans, and hydroids),  should be based  on the area covered by the "hold fast" (the 
attached base of the organisms) and not by the area covered by the "fronds" (leaves of 
macro-algae) or offshoot colonies. 

Overall  fouling assessment: The individual assessments of the fouling coverage  of  each 
type  of  organism  may  be  combined  to  provide  an  overall  fouling  assessment.  To 
generate  this,  a  weighting  of  the  coverage  of  the  different  types  of  fouling  may  be 
applied to rate and characterise the severity of the fouling present. 

When  the  coating  under  test  is  intended  for  use  on  ships,  fouling  never  seen  on  active 
vessels (e.g. amphipods) may be disregarded  during the weighting. Biofouling attached 
to other fouling organisms (secondary fouling) should also be excluded from the overall 
fouling assessment. 

Only  the  fully  immersed  surface  area  (if  parts  of  the  panel  are  subject  to  splash  only) 
should  be  included  in  the  determination  of  the  fouling  rating.  Fouling  attached  within  1 
cm from all edges of the test panel and fouling around the cable ties/studs/etc. may be 
disregarded  in  cases  where  an  edge  effect  is  seen.  (Fouling  around  edges  is  normally 
attributed to insufficient antifouling paint film thickness around sharp panel edges.) 

 Fouling caused by physical defects or damages in the substrate  or accidental damages 
of the antifouling should be disregarded. Fouling on exposed anticorrosive paints or other 
substrates  (except  where  these  are  used  as  negative  controls)  or  on  other  antifouling 
paints that may be used to coat panel edges, should be excluded from the assessment. 

Physical  defects  (detachment,  blistering,  cracking,  etc.)  attributed  to  the  inherent 
properties of the antifouling paint itself should be recorded and reported. 

Photos: Inspection reports should include panel photos from each inspection. 

7. 

Reporting 

The  report  should  contain  all  relevant  information  obtained  from  the  efficacy  trial  for  a 
given product. This may include: 

• 

• 

• 

• 

The  name  of  the  reporting  company  (and  client  if  the  test  is  carried  out  on 
assignment) 

The geographical location of the test raft(s) (including longitude and latitude) 

The geography (e.g. open sea, bay, estuary, etc.), depth of water, and water 
exchange conditions (tide, currents) at the raft site 

Typical  local  conditions.  E.g.  water  temperature,  salinity,  and  pH  at  the  raft 
site 

•  Relevant  information  on  the  typical  fouling  community  at  the  test  site  and 

seasonal influences where applicable. 

•  A  discussion  of  any  special  conditions  or  variables  that  may  have  arisen 

particular to the specific test 

•  Orientation and exposure depth of test panels 

•  Dimensions and type (material) of test panels 

• 

Identification of the tested product and control(s) 

•  Details on the panel preparation for the product under test and the control(s) 

(No. of coats, film thickness, application technique, etc.) 

•  Number of replicates if used 

• 

Initial  date  of  immersion  and  the  cumulative  exposure  time  (in  months)  for 
subsequent inspections 

•  Raw data from each individual assessment of a test panel 

 
 
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The overall fouling assessment rating at each inspection during the exposure 
period 

Photos of test and control panels 

•  A  systematic  appraisal  of  the  efficacy  of  the  test  product  in  relation  to  the 
negative control and, if used, any positive controls and the method by  which 
that appraisal has been conducted 

•  A  description  of  the  reporting  company's  weighting  system  used  to  provide 

the overall fouling assessment rating 

•  A discussion on the validity and acceptability of the test result relative to the 
intended  label  claim  for  the  product  tested  when  commercialised  [e.g. 
recommended  use  area  (recreational  yachts,  ships'  niche  areas,  ships'  flat 
bottoms, ships' water line, etc.) protection time/dry-docking interval,  fouling 
conditions in targeted markets, etc.]. 

An  interpretation  of  the  test  data  generated  and  a  conclusion  on  the  efficacy  of  the 
coating under test. 

 
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Appendix 22. Example  of  how  an  overall 
fouling 
assessment  may  be  carried  out  for  panel  testing  in 
marine waters 

In order to assess panels out in the field, an effective and simple system is needed. Very 
detailed assessments of fouling coverage do not increase the quality of the test,  as field 
conditions  are  highly  variable  and  static  raft  tests  can  only  provide  an  indication  of 
products’ real life performance. 

Individual  companies  have  different  ways  of  assessing  the  coverage  of  the  main 
categories  of  fouling  into  an  overall  description  of  the  efficacy  of  test  panels.  However, 
the principles of the example should apply to most assessment systems. Transparency of 
how  the  overall  assessment  is  carried  out  is  important  in  order  to  evaluate  an  efficacy 
report. 

The  fouling  coverage  on raft panels will be assessed based on coverage intervals. Each 
interval will be recorded by a different 'rating'. 

Table 51: Example of categorisation of fouling coverage into ratings from 0 to 4 

Fouling  Coverage  (examples  of  company  specific 
intervals for coverage of fouling) 

Rating 

Company 1 

Company 2  

0-10% 

10-30% 

30-50% 

50-80% 

80-100% 

0% 

>0-25% 

25-50% 

50-75% 

75-100% 

0 

1 

2 

3 

4 

As  different  fouling  species  can  contribute  to  different  impacts  on  a  vessel  (e.g.  fuel 
consumption  of  a  ship),  the  coverage  ratings  may  be  weighted  in  several  ways  to  take 
this  into  account.  The  applicant  may  provide  references  to  literature  that  provide  more 
detail on the assessment and weighting factors54. 

Table 52: Example of weighting of ratings 

Type of fouling 

Light slime 

Dense slime 

Macro-algae 

Animals 

Weighting (of ratings from 1-4) 

Trace (1) 

Slight (2) 

Medium (3)  Heavy (4) 

0 

3 

5 

5 

1 

5 

10 

10 

3 

10 

30 

30 

5 

20 

50 

50 

54 e.g. IMO MEPC/60/4/21, 2010 from IPPIC  

 
 
 
 
 
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A  score  may  be  calculated  by  adding  up  the  weightings.  In  this  example,  that  value  is 
then  subtracted  from  100.  Zero  growth  (apart  from  traces  of  light  slime)  gives  the 
fouling resistance rating 100 (100-0) and heavy fouling of both algae and animals gives 
the  rating  0  [100-(50+50)].  The  rating  is  then  allocated  to  descriptions  of  the  overall 
efficacy. 

Table 53: Example of categorisation of overall efficacy 

Fouling resistance rating 

Company specific score intervals, each with 
a  corresponding  characterisation  of  the 
efficacy 

Efficacy 

Excellent 

Good 

Fair 

Poor 

Description of types of fouling: 

Slime: 

Bacteria, micro-algae, and protozoa. 

Light slime is easily removed from the surface. 

Dense slime is not easily removed from the surface. 

Algae (weed):   Green algae, red algae, and brown algae. 

Animals: 

Barnacles, tubeworms, mussels, hydroids, and bryozoans. 

RELATING  COMPANY  FOULING  ASSSESSMENTS  TO  THE  NORMS  AND  CRITERIA  FOR 
PRODUCT AUTHORISATION. 

When  applying  for  authorisation  of  an  antifouling  product,  the  applicant  should  provide 
their  overall  fouling  assessment  of  the  product,  together  with  the  raw  data  and 
photographs/diagrams of the panel tests. 

This guidance document only takes into account the percentage of macro-fouling on the 
raft  panels  as  pass/fail  criterion,  not  the  classification  in  the  applicant’s  assessment 
system. 

As the percentage coverage per rating may differ between different company’s 
assessment systems (see Table 51), some systems might not record 25 % coverage (the 
pass/fail criterion) in their rating system (e.g. in Table 51 Company 1 has a borderline at 
30 % not at 25 %). Therefore, not only the ratings and end category of the product 
should be provided but also the raw data of the panel tests. The percentage coverage 
with macro-fouling per panel can then be identified from the raw data. This percentage is 
used to see if the product is sufficiently effective (i.e. <25 % macro-fouling). 

 
 
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477 

Appendix 23. PT  22  active  substances  in  the  review 
programme 

Table 54: PT 22 active substances in the review programme 

Active Substance 

Formaldehyde 

Bronopol 

Iodine 

Quaternary ammonium compounds, benzyl-C12-18-
alkyldimethyl, chlorides 

Quaternary ammonium compounds, benzyl-C 12- 16-
alkyldimethyl, chlorides (ADBAC) 

Quaternary ammonium compounds, benzyl-C12-14-
alkyldimethyl, chlorides 

Quaternary ammonium compounds, C12-14-
alkyl[(ethylphenyl)methyl]dimethyl, chlorides 

RMS 

CAS No 

DE 

ES 

SE 

IT 

IT 

IT 

IT 

50-00-0 

52-51-7 

7553-56-2 

68391-01-5 

68424-85-1 

85409-22-9 

85409-23-0 

Polyvinylpyrrolidone iodine 

SE 

25655-41-8 

 
 
 
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Appendix 24. Assessment grid for tests on human bodies 

This grid is for use in the assessment of the biocidal product itself, but not for assessing 
the overall embalming process with its hygiene and cosmetic aspects. 

Number of the report: 

Name and signature of the embalming professional:  

Company:  

Address of company: 

1.  General information 

Date of the operation:  

Place:  

Type of place: 

□ Funeral parlour 
□ Morgue 
□ Establishment without a morgue (fewer than 200 deaths per year) 
□ Home or other (please specify): 
Identification: 

Gender: □ Male □ Female 
Age: 

Estimated weight (kg): 

Estimated corpulence: □ cachectic □ thin □ medium □ stout 
Adiposity: □ low □ medium □ high 
Date of death (if known): 

Date and time of treatment: 

Body refrigerated: □ yes □ no. If "yes", for how long:  
Temperature: 

Causes of death (if known): 

Therapeutic treatment (if known): 

2.  Preoperative examination of the body 

Body intact: □ yes □ no, description:  

Autopsy before treatment: □ yes □ no 

Presence of external prostheses: □ yes □ no 

 
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Surgical intervention before death (if apparent or known): □ yes □ no 
If yes, type of intervention: 

Other visible anomaly(ies): 

Decomposition: □ none □ commencing □ problematic 

Rigidity: □ none □ minimal □ moderate □ problematic 

Dehydration: □ none □ normal □ high 

Lividity: □ none □ minimal □ moderate □ problematic, location:  

Coloration of tissues (yellowing): □ no □ slight □ moderate □ intense, description:  

Dermal lesions (sores, blisters, wounds, etc.): □ yes □ no, description: 

Distension of the abdomen: □ no, □ slight □ moderate □ intense, □ liquid □ gas 

Bruising: □ yes □ no, □ abdomen □ thorax □ leg, □ arm, □ face, specify degree and 
place: 

Comments: 

3.  Techniques used for injection of the biocidal product 

Time of start of treatment: 

Time of end of treatment: 

Site(s) of injection: 
Carotid(s): □ right □ left.  
Femoral(s): □ right □ left 
Axillary(ies): □ right □ left 
Other(s), description:  
Ease of finding: □ easy □ normal □ deep 
Condition: □ good □ atheromatous / □ hardened 
Injection: □ manual □ by electric pump □ by gravity 
Diffusion: □ good □ fair □ bad 
Puncture before treatment: □ yes □ no. If "yes", type: 
Biocidal product used: 

Pre-injection: □ yes □ no 
Injection:  

Hypodermic:  

product: 

 
 
 
 
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Site: 

product: 

Site: 

480 

Topical: 

Name of the biocidal product:  

Active substance(s): 

Duration of efficacy claimed: 

Number of litres: 

Arterial fluid:  

Name of fluid: 

% of dilution:  

Number of litres injected:  

Start time for the injection: 

End time for the injection: 

Cavity treatment:  

Name of fluid:  

% of dilution: 

Number of litres injected:  

Start time for the injection: 

End time for the injection: 

Corrective injection:  

□ yes □ no 
Drainage method: 

□ Cardiac, □ Venous 
Vein(s) chosen: □ jugular, □ femoral, □ axillary 
Volume drained by circulatory system (litres):  

Total volume drained (litres): 

Type of drainage: □ drain tube(s) □ forceps □ intermittent / □ continual 
Quality of drainage: □ considerable clotting □ medium □ slight □ no clotting 
General puncture: 

Quantity: 

 
 
 
 
 
 
 
 
 
 
 
 
  
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4.  Observations concerning the injection of the biocidal product 

Observations during the treatment: 

Odour: □ normal □ fair □ bad 

Colouring: □ good □ fair □ bad 

Suppleness of the skin: □ good □ fair □ bad 
Observations following the treatment: 

Odour: □ good □ fair □ bad 

Colouring: □ good □ fair □ bad 

Suppleness of the skin: □ good □ fair □ bad 
Mandatory observation 48 hours after the treatment: 

Odour: □ good □ fair □ bad 

Colouring: □ good □ fair □ bad 

Suppleness of the skin: □ good □ fair □ bad 
Optional observation (at times relevant to the manufacturer's claims): 

Time after treatment: 

Odour: □ good □ fair □ bad 

Colouring: □ good □ fair □ bad 

Suppleness of the skin: □ good □ fair □ bad 
Other products used during the preservation process: 

Reasons for their use:  

Description: 

Products for cosmetic purposes:  

□ yes, □ no. If yes: □ normal, □ make-up, □ significant, □ restorative  
Other restoration: ___________________ 

Moisteners  and  other  products  used  (cauterising  agents,  disinfectants,  skin  tone 
correctors, etc.):  

Name of the fluid: _____________________, % dilution: ______, litres injected: ___ 

Name of the fluid: _____________________, % dilution: ______, litres injected: ___ 

Explanations:  

5.  Comments 

 
 
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Version 4.0 December 2021   

EUROPEAN CHEMICALS AGENCY 
P.O. Box 400, 
FI-00121 Helsinki, Finland 
echa.europa.eu