Guidance on the Drinking Water Directive. Volume II, Methodologies for accepting starting substances, compositions and constituents for use in the manufacture of materials or products in contact with water intended for human consumption.
Date: 2025-05-09
Institutions: European Chemicals Agency
Subjects: biocide, chemical product, chemicals legislation, consumer protection, dangerous substance, directive (EU), drinking water, information analysis, public health, risk management

G U I D A N C E  

Guidance on the Drinking Water Directive 

Volume II: Methodologies for accepting starting 
substances, compositions and constituents for use in the 
manufacture of materials or products in contact with water 
intended for human consumption 

Version 1.0 

 
 
 
 
 
 
 
 
 
 
Guidance on the DWD: Volume II 
Version 1.0 – January 2025 

Legal notice 

This  document  aims  to  assist  users  in  complying  with  their  obligations  under  Directive  (EU) 
2020/2184 of the European Parliament and of the Council of 16 December 2020 on the quality 
of water intended for human consumption (recast), hereafter referred to as the drinking water 
directive (DWD). However, users are reminded that the text of the DWD (Article 11(2)(a)) and 
Commission Implementing Decision (EU) 2024/365 are the only authentic legal references and 
that the information in this document does not constitute legal advice. Usage of the information 
remains 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 Drinking Water Directive – Volume II: Methodologies for accepting 
starting substances, compositions and constituents for use in the manufacture of 
materials or products in contact with water intended for human consumption 

ECHA-24-G-12-EN  

Reference: 
Cat. number:  ED-01-24-023-EN-N 
978-92-9468-446-2 
ISBN: 
10.2823/6310589 
DOI: 
January 2025 
Publ. date: 
EN 
Language: 

© European Chemicals Agency, 2025 

Luxembourg: Publications Office of the European Union, 2025 

If you have questions or comments in relation to this document, please send them (quoting 
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 DWD: Volume II 
Version 1.0 – January 2025 

3 

Preface 

Guidance on the Drinking Water Directive – Volume II (accepting methodology), is to be used in 
preparing  applications  for  starting  substances,  compositions  and  constituents  for  approval  for 
inclusion in the European positive lists. 

This  document  describes  the  applicants’  obligations  according  to  Article 11(2)(a)  of  Directive 
(EU) 2020/2184 and how to fulfil them. 

The guidance provides technical scientific advice on how to comply with the methodologies for 
testing and how to perform the risk assessment and the exposure assessment for the evaluation 
of human health set out in Commission Implementing Decision (EU) 2024/365. 

Notes for the reader 

This  guidance  must  be  read  in  conjunction  with  the  relevant  DWD  implementing 
legislation, particularly: 

•  Commission Implementing Decision (EU) 2024/365 of 23 January 2024 laying down 
rules for the application of Directive (EU) 2020/2184 of the European Parliament and 
of the Council as regards methodologies for testing and accepting starting substances, 
compositions and constituents to be included in the European positive lists; 

•  Commission Implementing Decision (EU) 2024/367 of 23 January 2024 laying down 
rules for the application of Directive (EU) 2020/2184 of the European Parliament and 
of  the  Council  by  establishing  the  European  positive  lists  of  starting  substances, 
compositions and constituents authorised for use in the manufacture of materials or 
products that come into contact with water intended for human consumption; 

•  Commission Delegated Regulation (EU) 2024/369 of 23 January 2024 supplementing 
Directive  (EU)  2020/2184  of  the  European  Parliament  and  of  the  Council  by  laying 
down the procedure regarding inclusion in or removal from the European positive lists 
of starting substances, compositions and constituents. 

In  addition,  this  guidance  document  is  part  of  wider  guidance  on  DWD  applications  
comprising a total of five volumes. The remaining volumes are: 

•  DWD  Guidance  Volume I  on  methodologies  for  testing  starting  substances, 
compositions and constituents for use in the manufacture of materials or products in 
contact with water intended for human consumption; 

•  DWD Guidance Volume III on the scope of DWD applications; 
•  DWD Guidance Volume IV on the contents of a notification of intention; 
•  DWD  Guidance  Volume V  on  calibrants  and  representative  samples  of  accepted 
compositions  in  connection  to  applications  submitted  under  Commission  Delegated 
Regulation (EU) 2024/369 (this guidance was prepared by the European Commission 
Joint Research Centre). 

 
 
 
 
 
 
 
4 

Contents 

Guidance on the DWD: Volume II 
Version 1.0 – January 2025 

Legal notice .................................................................................................... 2 

Preface ........................................................................................................... 3 

Contents .......................................................................................................... 4 

List of figures .................................................................................................... 8 

List of tables ..................................................................................................... 9 

Abbreviations .................................................................................................... 10 

1.  Introduction .................................................................................................. 12 
1.1.  Evaluation ............................................................................................ 12 
1.2.  Assessment .......................................................................................... 13 

2.  General principles .......................................................................................... 15 

3.  Hazard assessment: hazard identification ......................................................... 17 
3.1.  Collection and evaluation of all available data ............................................ 21 
3.1.1.  Collection of data (step 4 in Figure 1 and Figure 2) ............................ 21 
3.1.2.  Evaluation of the data (step 5 in Figure 1 and Figure 2) ...................... 21 
3.1.2.1.  Completeness of data ................................................................. 21 
3.1.2.2.  Adequacy of data ....................................................................... 21 
3.1.2.2.1.  Reliability of data ................................................................... 22 
3.1.2.2.2.  Relevance of data .................................................................. 23 
3.1.2.2.3.  Human data .......................................................................... 24 
In vitro data .......................................................................... 26 
3.1.2.2.4. 
3.1.2.2.5. 
(Quantitative) structure–activity relationships ........................... 26 
3.1.3.  Read-across approach .................................................................... 31 
3.1.4.  Weight of evidence (WoE) ............................................................... 32 
3.2.  Toxicokinetics ....................................................................................... 33 
3.2.1.  Definitions and uses of toxicokinetic studies ...................................... 33 
3.2.2.  Main principles of toxicokinetic studies ............................................. 34 
3.2.2.1.  Accumulation ............................................................................ 35 
3.2.3.  Prediction of toxicokinetics .............................................................. 35 
3.2.3.1.  Absorption ................................................................................ 35 
3.2.3.2.  Distribution ............................................................................... 38 
3.2.4.  Accumulation potential ................................................................... 39 
3.2.4.1.  Metabolism ............................................................................... 40 
3.2.4.2.  Excretion .................................................................................. 40 
3.2.5.  Generating and integrating toxicokinetic information .......................... 42 
3.2.6.  Other methods for generating absorption, distribution, metabolism and excretion 
data ............................................................................................. 42 
3.2.6.1.  In vitro studies .......................................................................... 42 
3.2.6.2.  In silico methods: kinetic modelling ............................................. 43 
3.2.7.  Variability and uncertainty in toxicokinetics ....................................... 43 
3.2.8.  Metabolism studies as a basis for internal dose considerations ............. 43 
3.2.9.  Extrapolation ................................................................................. 45 
3.2.9.1.  Interspecies extrapolation ........................................................... 46 
3.2.9.2.  Interroute extrapolation .............................................................. 47 
3.3.  Repeated dose toxicity ........................................................................... 48 
3.3.1.  Definition of repeated dose toxicity .................................................. 48 
3.3.2.  Data to be used in the hazard assessment ........................................ 49 

 
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3.3.2.1.  Non-human data for repeated dose toxicity ................................... 49 
3.3.2.1.1.  Non-testing data for repeated dose toxicity ............................... 49 
3.3.2.1.2.  Testing data for repeated dose toxicity ..................................... 49 
3.3.3.  Specific system/organ toxicity ......................................................... 59 
3.3.3.1.  General aspects ......................................................................... 59 
3.3.3.2.  Neurotoxicity ............................................................................. 59 
3.3.3.2.1.  Definition of neurotoxicity ....................................................... 59 
3.3.3.2.2. 
Introduction .......................................................................... 59 
3.3.3.2.3.  Structure–activity considerations ............................................. 60 
3.3.3.2.4.  Assessment of available information or results from initial testing  60 
3.3.3.2.5.  Further neurotoxicity testing ................................................... 61 
3.3.3.3.  Immunotoxicity ......................................................................... 61 
3.3.3.3.1.  Definition of immunotoxicity .................................................... 61 
Introduction .......................................................................... 61 
3.3.3.3.2. 
3.3.3.3.3. 
Immunosuppression ............................................................... 62 
3.3.3.4.  Effects on the endocrine system .................................................. 64 
3.3.3.5.  Remaining uncertainty ................................................................ 64 
3.3.3.5.1.  Threshold of the critical effect.................................................. 65 
3.3.3.5.2.  Other considerations .............................................................. 65 
3.3.4.  Conclusions on repeated dose toxicity .............................................. 65 
3.3.5.  Concluding on suitability for risk assessment ..................................... 66 
3.4.  Mutagenicity ......................................................................................... 67 
3.4.1.  Definition ...................................................................................... 67 
3.4.2.  Data to be used in the assessment of genotoxicity/mutagenicity .......... 68 
3.4.2.1.  Non-human data for mutagenicity ................................................ 68 
3.4.2.1.1.  Non-testing data for mutagenicity ............................................ 68 
3.4.2.1.2.  Testing data for mutagenicity .................................................. 68 
3.4.2.2.  Human data .............................................................................. 73 
3.4.3.  Remaining uncertainty on mutagenicity ............................................ 73 
3.4.4.  Concluding on suitability for risk assessment ..................................... 74 
3.4.4.1.  Considerations on dose–response shapes and mode of action of mutagenic 

substances in test systems ............................................................. 74 
3.5.  Carcinogenicity ...................................................................................... 75 
3.5.1.  Definition ...................................................................................... 75 
3.5.2.  Data to be used in the hazard assessment ........................................ 76 
3.5.2.1.  Non-human data for carcinogenicity ............................................. 76 
3.5.2.1.1.  Non-testing data for carcinogenicity ......................................... 76 
3.5.2.1.2.  Testing data for carcinogenicity ............................................... 77 
3.5.2.2.  Human data .............................................................................. 82 
3.5.3.  Remaining uncertainty .................................................................... 82 
3.5.4.  Concluding on suitability for risk assessment ..................................... 83 
3.6.  Reproductive toxicity .............................................................................. 84 
3.6.1.  Definition ...................................................................................... 84 
3.6.2.  Data to be used in the effects assessment ........................................ 85 
3.6.2.1.  Non-human data ........................................................................ 85 
3.6.2.1.1.  Non-testing data .................................................................... 85 
3.6.2.1.2.  Testing data for reproductive toxicity ....................................... 85 
3.6.2.1.3.  Relation between maternal toxicity and developmental toxicity .... 91 
3.6.2.1.4.  Reproductive toxicity via lactation ............................................ 92 
3.6.2.2.  Human data .............................................................................. 93 
3.6.3.  Remaining uncertainty on reproductive toxicity.................................. 93 
3.6.4.  Conclusions on reproductive toxicity ................................................. 93 
3.6.5.  Concluding on suitability for risk assessment ..................................... 93 

 
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Guidance on the DWD: Volume II 
Version 1.0 – January 2025 

4.  Hazard assessment: hazard characterisation (dose–response / concentration-response 
relationship) ..................................................................................................... 94 
4.1.  Introduction .......................................................................................... 94 
4.2.  Identification of critical effects ................................................................. 96 
4.2.1.  Hazard information underlying the derivation of a derived no-effect level ... 96 
4.2.1.1. Toxicokinetics and absorption .............................................................. 96 
4.2.1.2. Systemic (target organ) toxicity after repeated dose administration ......... 96 
4.2.1.3. Toxicity to reproduction and development ............................................. 97 
4.2.1.4. Other toxicity end points .................................................................... 97 
4.2.2.  Derived no-effect level derivation for threshold effects ............................. 97 
4.2.2.1. Relevant time frames in DNEL derivation .............................................. 98 
4.2.2.2. Application of appropriate assessment factors ....................................... 98 
4.2.2.3. Intraspecies differences ...................................................................... 100 
4.2.3.  Dose–response assessment .................................................................. 102 
4.3.  Non-threshold effects ............................................................................. 102 

5.  Exposure assessment ..................................................................................... 103 
5.1.  Determination of concentration at the tap from physical testing .................. 103 
5.1.1.  Organic materials ................................................................................ 103 
5.1.2.  Metallic materials ................................................................................. 104 
5.1.2.1. Determination of concentration at the tap values from physical testing in accordance 
with EN 15664-1 ................................................................................. 104 
5.1.3.  Cementitious materials ......................................................................... 105 
5.1.4.  Enamel, ceramic and other inorganic materials ........................................ 105 
5.1.5.  Surface layers ..................................................................................... 106 
5.2.  Determination of concentration at the tap from modelling for organic materials 

107 

6.  Risk acceptance............................................................................................. 108 
6.1.  Main principles ...................................................................................... 108 
6.1.1.  Approaches to maximum tolerable concentration at the tap adoption for relevant 
chemical species .................................................................................. 108 
6.1.2.  Concentration at the tap and migration tiers ........................................... 108 
6.2.  Limited approach to the adoption of a maximum tolerable concentration at the tap value

  109 

6.2.1.  Limited approach 1: adoption of a maximum tolerable concentration at the tap value 
on the basis of a robust past assessment ................................................ 109 

6.2.2.  Limited approach 2: maximum tolerable concentration at the tap value for relevant 
chemical species classified as carcinogenic, mutagenic or toxic to reproduction 
category 1A or 1B / endocrine disruptor human health category 1/PBT/vPvB/PMT/vPvM
  112 

6.2.3.  Limited approach 3: maximum tolerable concentration at the tap value for relevant 

chemical species that are potential carcinogens, mutagens or endocrine disruptors with 
a non-threshold mode of action ............................................................. 114 

6.2.4.  Limited approach 4: maximum tolerable concentration at the tap value for relevant 

chemical species for which available toxicological information is insufficient  114 
6.3.  Comprehensive approach to the adoption of a maximum tolerable concentration at the 
tap value on the basis of toxicological information ................................... 118 

 
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6.3.1.  Maximum tolerable concentration at the tap for a relevant chemical species of low 

migration potential .............................................................................. 118 
6.3.2.  Maximum tolerable concentration at the tap for a relevant chemical species of medium 
migration potential .............................................................................. 118 

6.3.3.  Maximum tolerable concentration at the tap for a relevant chemical species of high 

migration potential .............................................................................. 119 
6.4.  Selection of allocation factors .................................................................. 120 
6.5.  Risk acceptance methodology.................................................................. 121 
6.5.1.  Standard methodology ......................................................................... 121 
6.5.2.  Metallic compositions ........................................................................... 121 
6.5.2.1. Metallic compositions tested according to EN 15664-1 ............................ 122 
6.5.2.2. Metallic compositions tested according to EN 16056 (passivating alloys) ... 123 
6.5.3.  Surface layers (coatings, platings and linings) ......................................... 124 

7.  References ................................................................................................... 125 

8.  Annex I – Schematic of the metallics acceptance process ................................... 133 

 
 
 
 
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List of figures 

Guidance on the DWD: Volume II 
Version 1.0 – January 2025 

Figure 1. Schematic representation of the stepwise approach for fulfilling the testing 
methodology for human health hazard for starting substances and constituents, for the 
purpose of the DWD .......................................................................................... 19 
Figure 2. Schematic representation of the stepwise approach for fulfilling the testing 
methodology for human health hazard for compositions, for the purpose of the DWD  20 
Figure 3. Use of increasing knowledge on substance metabolism ............................. 44 
Figure 4. Schematic representation of the steps for performing the hazard assessment  95 

 
 
 
 
 
Guidance on the DWD: Volume II 
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List of tables 

9 

Table 1. How different types of data can be used to interpret oral/GI absorption ....... 37 
Table 2. How different types of data can be used to interpret substance distribution .. 39 
Table 3. How data from different sites can be used to interpret accumulation after oral 
exposure .......................................................................................................... 40 
Table 4. How physico-chemical data can affect excretion after oral exposure ............ 41 
Table 5. Overview of in vivo oral repeated dose toxicity test guideline studies ........... 54 
Table 6. Overview of other in vivo test guideline studies giving information on repeated dose 
toxicity ............................................................................................................. 57 
Table 7. In vitro test methods ............................................................................. 69 
Table 8. Somatic cells – in vivo test methods ........................................................ 70 
Table 9. Germ cells – in vivo test methods............................................................ 71 
Table 10. Overview of in vivo OECD test guidelines for reproductive toxicity ............. 89 
Table 11. Allometric scaling factors for different animal species when compared with humans 
(assuming the human body weight is 70 kg) ......................................................... 99 
Table 12. Migration tiers equivalents for FCM and DWCM assessments ..................... 109 
Table 13. Sources of existing MTCtap values for different material types .................... 110 
Table 14. Summary of limited risk acceptance approaches ...................................... 116 
Table 15. MTCtap values from toxicological data according to different migration tiers under the 
comprehensive approach .................................................................................... 120 

 
 
 
 
10 

Abbreviations 

ADME 

AF 

ALF 

AUC 

BMD 

Guidance on the DWD: Volume II 
Version 1.0 – January 2025 

absorption, distribution, metabolism and excretion 

assessment factor 

allocation factor 

area under the curve 

benchmark dose 

CLP regulation 

Regulation (EC) No 1272/2008 on classification, labelling and packaging of 
substances and mixtures  

CMR 

CNS 

Ctap 

DNEL 

DTU 

DWCM 

DWD 

ECETOC 

ECHA 

ECVAM 

ED 

EEA 

EFSA 

EUPL 

FCM 

GI 

GLP 

IPCS 

JRC 

LOAEC 

LOAEL 

Kow 

KP,W 

MEPa(T) 

carcinogenic, mutagenic or toxic to reproduction 

central nervous system 

concentration at the tap 

derived no-effect level 

Technical University of Denmark 

drinking water contact material 

drinking water directive 

European Centre for Ecotoxicology and Toxicology of Chemicals 

European Chemicals Agency 

European Centre for the Validation of Alternative Methods 

endocrine disruptor 

European Union + Norway, Iceland and Liechtenstein 

European Food Safety Authority 

European positive list 

food contact material 

gastrointestinal 

good laboratory practice 

International Programme on Chemical Safety 

Joint Research Centre 

lowest observed adverse effect concentration 

lowest observed adverse effect level 

partition coefficient octanol–water 

partition coefficient organic material-water 

average of the arithmetic mean of the equivalent pipe concentrations 
(MEPn(T)) of the three test lines in one rig 

 
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11 

MTCtap 

maximum tolerable concentration at the tap 

N(L)OAEL 

NOAEL and/or LOAEL 

NOAEC 

NOAEL 

OECD 

PBPK 

PBT 

PMT 

no observed adverse effect concentration 

no observed adverse effect level 

Organisation for Economic Co-operation and Development 

physiologically based pharmacokinetic 

persistent, bioaccumulative and toxic 

persistent, mobile and toxic 

(Q)SAR 

(quantitative) structure–activity relationship 

REACH regulation 

Regulation (EC) No 1907/2006 on the registration, evaluation, authorisation 
and restriction of chemicals 

SML 

TG 

TK 

TOC 

specific migration limit 

Test Guideline 

toxicokinetic 

total organic carbon 

US EPA 

United States Environmental Protection Agency 

vPvB 

vPvM 

w/w 

WoE 

very persistent and very bioaccumulative 

very persistent and very mobile 

weight per weight ratio 

weight of evidence 

 
 
 
12 

1. 

Introduction 

1.1.  Evaluation 

Guidance on the DWD: Volume II 
Version 1.0 – January 2025 

To ensure a high level of protection of human health, any risks arising from relevant chemical 
species  that  migrate  into  water  intended  for  human  consumption  as  a  result  of  the  use  of  a 
starting substance, composition or constituent must be identified. 

This document provides technical and scientific advice on how to fulfil the common principles set 
out  in  Annex VI  to  Commission  Implementing  Decision  (EU)  2024/365  of  the  acceptance 
methodology for starting substances, compositions and constituents, based on the assessment 
of the risk raised by the relevant chemical species. 

According to Section 2 of Annex VI to Commission Implementing Decision (EU) 2024/365, the 
acceptance methodology should be based on risk assessment that shall result in: 

•  determining the maximum tolerable concentration at tap water (MTCtap) for each relevant chemical 

species; 

•  ensuring that Ctap [the concentration at the tap] for each relevant chemical species is lower than 

its MTCtap. 

In addition to the information required under Annexes I, II and III, a risk assessment shall take into 
account  any  other  relevant  technical  or  scientific  information  which  is  available  addressing  worst 
foreseeable conditions of use. Where appropriate, conditions of use shall be implemented. 

The information provided in the risk assessment shall allow the Committee for Risk Assessment of ECHA 
to  evaluate  and  reach  an  opinion  on  whether  the  starting  substance,  composition  or  constituent 
complies with the criteria set out under Article 11(1) of Directive (EU) 2020/2184. 

This  guidance  is  primarily  addressed  to  applicants  seeking  approval  for  a  starting  substance, 
composition  or  constituent  to  be  included  in  a  European  positive  list  (EUPL)  and  also  those 
seeking the removal or updating of an existing entry in an EUPL. 

This  guidance  needs  to  be  considered  together  with  Annex V  (‘Toxicological  properties’)  to 
Commission  Implementing  Decision  (EU)  2024/365  and  Guidance  on  the  Drinking  Water 
Directive – Volume I, hereafter DWD Guidance Volume I, on testing methodologies. 

In this document, unless explicitly described otherwise, the term ‘applied-for entry’ is used to 
refer  to  starting  substances,  compositions  and  constituents  for  which  applicants  may  seek 
approval for inclusion in or removal from an EUPL, or their renewal, from the European Chemicals 
Agency  (ECHA).  This  is  how  the  starting  substance,  composition  or  organic  cementitious 
constituent  would  be  presented  if  included  in  an  EUPL.  In  contrast,  for  the  purpose  of 
toxicological  properties  assessment  and  risk  assessment  and  acceptance,  the  term  ‘relevant 
chemical  species’,  as  identified  in  accordance  with  Section 3  of  Annex IV  to  Commission 
Implementing Decision (EU) 2024/365, is used. 

A glossary of key terms, also relevant to this guidance document, is provided in DWD Guidance 
Volume I. 

 
 
 
Guidance on the DWD: Volume II 
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1.2.  Assessment 

13 

The risk assessment process in relation to human health entails a sequence of actions outlined 
below. 

•  Hazard assessment, comprising the following. 

o  Hazard  identification.  Identification  of  the  adverse  effects  that  relevant  chemical 

species have an inherent capacity to cause. 

o  Hazard characterisation. Dose (concentration)–response (effects) assessment, which 
is  the  estimation  of  the  relationship  between  dose,  or  level  of  exposure  to  relevant 
chemical species, and the incidence and severity of an effect, where appropriate. Based 
on this information, a threshold concentration called maximum tolerable concentration at 
the tap (MTCtap) should be calculated for each relevant chemical species. 

•  Exposure assessment. Estimation of the concentrations/doses to which human populations 
(i.e.  consumers)  are  or  may  be  exposed.  In  the  context  of  the  drinking  water  directive 
(DWD),  this  is  to  establish  the  concentration  at  the  tap  (Ctap),  that  is,  the  value  that 
constitutes the amount of the relevant chemical species in drinking water to which humans 
are potentially exposed. 

•  Risk  acceptance.  In  the  context  of  the  DWD,  the  applied-for  entry  does  not  pose  an 
unacceptable risk if, for all relevant chemical species, the Ctap is lower than the corresponding 
MTCtap. 

A risk assessment covering all of the steps identified above must be carried out for all relevant 
chemical  species,  unless  they  meet  one  of  the  criteria  listed  in  Section 1  of  Annex VI  to 
Commission Implementing Decision (EU) 2024/365. For such substances, the first step – hazard 
assessment – is omitted (see also Section 6.1.1 of DWD Guidance Volume I and Sections 6.2.1–
6.2.4 of this document). 

The risk assessment for human health must address the following potential toxic effects and the 
exposure of the general human population by the oral route: 

• 

systemic (target organ) toxicity after repeated dose administration; 

•  mutagenicity; 

• 

• 

toxicity for reproduction; 

carcinogenicity; 

•  neurotoxicity; 

• 

immunotoxicity; 

•  endocrine disruption. 

The  risk  assessment  should  be  carried  out  based  on  all  data  available,  applying  the  methods 
described in the following sections of this guidance. 

Applicants should note that applications under the DWD cannot be made for uses of chemical 
substances  that  would  result  in  the  weakening  of  a  restriction  under  Annex XVII  to  the 

 
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Guidance on the DWD: Volume II 
Version 1.0 – January 2025 

registration,  evaluation,  authorisation  and  restriction  of  chemicals  (REACH  regulation)  or  the 
authorisation regime of the REACH regulation. 

Note for the reader: 

The  text  of  Commission  Implementing  Decision  (EU)  2024/365  can  be  found  at 
http://data.europa.eu/eli/dec_impl/2024/365/oj. 

The term ‘chapter’ is used to refer to the highest level parts of the document (Chapter 1, 
2, 3, etc.). Lower level headings (e.g. 1.1) are referred to as ‘sections’. 

 
 
 
 
 
Guidance on the DWD: Volume II 
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2.  General principles 

15 

In the context of the DWD, the assessment should be based on the risks arising from all relevant 
chemical species. 

Section 3  of  Annex IV  to  Commission  Implementing  Decision  (EU)  2024/365  defines  relevant 
chemical species as follows. 

Relevant chemical species are those that are covered by the requirements set out in Annex V in order 
to demonstrate that the starting substance, composition or organic cementitious constituent meets the 
acceptance criteria set out in Annex VI. Relevant chemical species include the following: 

(a)  starting substances and organic cementitious constituents which function as a monomer or other 
reactant of a main polymer in the material; 

(b)  starting  substances,  organic  cementitious  constituents,  substance  constituents  and  non-
intentionally added species originating from the starting substance or organic cementitious constituent 
which  show  one  of  the human  health  hazards  referred  to  in  Section 1.1.  of  Annex VI  irrespective  of 
their levels of migration; 

(c)  starting  substances,  organic  cementitious  constituents,  substance  constituents  and  non-
intentionally added species originating from a starting substance or organic cementitious constituent 
which do not fall under points (a) or (b) and which have been tested in accordance with Table 1 [of the 
legal  act]  and  have  been  found  to  migrate  into  water  intended  for  human  consumption  with  a 
concentration at the tap (Ctap) exceeding 0,1 µg/l; 

(d)  metallic composition constituents or impurities, which have been tested in accordance with Table 1 
[of the legal act]; 

(e)  enamel, ceramic or other inorganic composition constituents or impurities of an enamel, ceramic 
or other inorganic composition which have been tested in accordance with Table 1 [of the legal act]. 

Based on the Ctap of a relevant chemical species in the water intended for human consumption, 
the applicant must submit toxicological information, as required under Section 2 of Annex VI to 
Commission Implementing Decision (EU) 2024/365. This information, together with any other 
relevant  technical  or  scientific  information  that  is  available,  must  be  considered  in  order  to 
perform a robust risk assessment. 

In  the  context  of  the  DWD,  the  procedure  for  the  human  health  risk  assessment  of  relevant 
chemical  species  consists  of  comparing  the  Ctap  derived  based  on  the  migration  studies 
(‘exposure’) with the MTCtap, an estimate of the amount of a substance in drinking water that 
can be ingested daily over a lifetime without appreciable health risk. For threshold effects, the 
MTCtap  is  based  on  the  derived  no-effect  level  (DNEL),  which  is  calculated  on  the  basis  of 
threshold levels such as the benchmark dose (BMD), no observed adverse effect level (NOAEL), 
lowest  observed  adverse  effect  level  (LOAEL)  and  no  observed  adverse  effect  concentration 
(NOAEC), with the use  of assessment factors (AFs), and which  constitute the outcome of the 
hazard characterisation. The NOAEL and/or LOAEL (N(L)OAEL) values are determined based on 
results from animal testing or on the basis of available human data. For non-threshold effects 
(e.g.  for  mutagenicity  or  carcinogenicity),  a  DNEL  cannot  be  derived.  For  relevant  chemical 
species exerting toxicity with a non-threshold mode of action, an MTCtap of 0.1 µg/l should be 
applied (further explanation is provided in Section 6.3). 

The derivation and use of dose–response relationships for each of the effects to be assessed are 
discussed in detail in Chapter 3 of this guidance document. 

 
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In their risk assessment, an applicant must identify any significant uncertainty (e.g. uncertainties 
related to the selection of the most relevant critical toxicological effect or to an allocation factor 
(ALF))  and  adequately  address  them  (e.g.  by  applying  a  reasonable  worst-case  approach)  if 
needed to ensure the protection of human health. 

For both the hazard assessment and exposure assessment, data on physico-chemical properties, 
including chemical reactivity, are needed. These data are required, for example, to accurately 
design and assess both the migration tests and toxicity studies. Physico-chemical properties may 
also provide indications of the absorption of the relevant chemical species after exposure via the 
oral route (the relevant route for drinking water). Chemical reactivity may be of importance, for 
example  in  the  estimation  of  the  exposure to  the  relevant  chemical  species,  and  also  has  an 
impact on its toxicokinetics and its metabolism. Information on the migration of the applied-for 
entry, the identification of the relevant chemical species and their migration concentrations into 
the test water is also needed, because it is used to determine the toxicological requirements for 
each relevant chemical species, as explained in DWD Guidance Volume I and according to the 
principles set out in Section 2 of Annex V to Commission Implementing Decision (EU) 2024/365. 

It  is  the  responsibility  of  the  applicant  to  secure  any  necessary  rights  to  use  any  third-party 
intellectual  property  in  their  application.  As  set  out  in  the  terms  and  conditions  of  the  ECHA 
submission tool ( 1), when submitting the application, the applicant is required to warrant that 
they  have  obtained  all  the  necessary  permission,  rights  or  licences  to  use  the  third-party 
intellectual property for the purpose of their application. The fact that a study or any other third-
party material is available on ECHA’s website does not mean that this material is not subject to 
the  third-party’s  intellectual  property  rights  or  that  it  may  be  freely  used  without  their 
permission. 

(1)  The tool will be available on ECHA’s website. 

 
 
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3.  Hazard assessment: hazard identification 

The principles of hazard assessment are stated in Part 2 of Section 2 of Annex VI to Commission 
Implementing Decision (EU) 2024/365. 

For each relevant chemical species, the hazard assessment process in relation to human health 
must entail assessment of effects, comprising the following. 

•  Hazard identification. The aim of hazard identification is to identify the effects of concern 

that a relevant chemical species has an inherent capacity to cause. 

•  Hazard characterisation: dose (concentration)–response (effect) assessment. This 
is the estimation of the relationship between dose, or level of exposure to a substance, and 
the incidence and severity of an effect. In this chapter it is referred to as ‘dose–response’. 
At this step the NOAEL, or, where it is considered more appropriate, the LOAEL or the BMD, 
is determined based on the observed effects, and a DNEL is derived. If appropriate, the shape 
of the dose–response curve should also be considered. 

The conditions under which hazard assessment does not apply to a relevant chemical species 
are stated under Section 1 of Annex VI to Commission Implementing Decision (EU) 2024/365. 

During both steps of the hazard assessment, it is very important to evaluate the available data 
for  adequacy  and  completeness.  The  evaluation  of  adequacy  must  address  the  reliability  and 
relevance of the data. 

For the effects of concern for which it is not possible to determine a N(L)OAEL, it is generally 
sufficient to evaluate whether the relevant chemical species has an inherent capacity to cause 
such an effect. Where for such an effect it is possible to draw a relationship between the dose 
or  concentration  of  the  relevant  chemical  species  and  the  severity  of  an  adverse  effect,  this 
relationship should be determined. 

Human data are in principle the most relevant source of information on human toxicity; however, 
since there may be limitations in terms of the reliability of human studies (see Section 3.1.2.2.3), 
they  are  normally  considered  together  with  animal  and  other  data.  Studies  conducted  with 
human volunteers are problematic from an ethical point of view. Results from such studies should 
be used only in justified cases (e.g. tests that were conducted for the authorisation of a medicinal 
product or when effects in already available human volunteer studies of existing substances have 
been  observed  to  be  more  severe  than  deduced  from  prior  animal  testing).  The  potential 
differences in sensitivity of human studies and studies in animals should be taken into account 
in  the  hazard  assessment,  on  a  case-by-case  basis.  In  relation  to  hazard  identification,  the 
relative  lack  of  sensitivity  of  human  data  may  cause  particular  difficulty:  negative  data  from 
studies in humans will not usually be used to override the classification of substances that have 
been classified on the basis of data from studies in animals in accordance with the criteria given 
in  Regulation  (EC)  No 1272/2008  on  classification,  labelling  and  packaging  of  substances  and 
mixtures (the CLP regulation), unless the classification is based on an effect that clearly would 
not be expected to occur in humans. For more information, see Guidance on the Application of 
the CLP Criteria ( 2). 

(2)  Guidance on the Application of the CLP Criteria Overview. 

 
 
 
 
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The structure of the section on hazard identification for each end point is as follows: 

•  definition of the effect of concern; 

•  data to be used in the hazard assessment; 

• 

• 

remaining uncertainty; 

conclusion on risk assessment. 

For  hazard  identification,  DWD  Guidance  Volume I  needs  to  be  considered  together  with  this 
guidance  and  that  in  Introductory  Guidance  on  the  CLP  Regulation ( 3).  The  CLP  regulation, 
Regulation  (EC)  No 1272/2008 (4),  is  an  umbrella  for  assessing  hazards  under  EU  chemical 
legislation and provides general rules that are relevant for hazard assessment under the DWD 
(e.g.  a  substance  is  classified  as  carcinogenic  unless  there  is  ‘strong  evidence’  that  the 
mechanism is not relevant for humans (CLP regulation, Annex I, Section 3.6.1.1)). It must be 
noted, however, that the hazard assessment under the DWD does not aim to classify substances 
according to CLP regulation criteria. 

For  the  purpose  of  hazard  assessment,  the  stepwise  approach  summarised  in  Figure 1  (for 
starting substances and organic cementitious constituents) and Figure 2 (for compositions) has 
to be followed. Steps 1–3 are taken first to identify if hazard assessment is needed. If it is, then 
steps 4 and 5 have to be followed. They include the collection of all available information and its 
assessment before deciding if additional testing needs to be performed. Once new test results 
become  available,  as  part  of  step 6  using  DWD  Guidance  Volume I,  these  results  should  be 
evaluated in accordance with the guidance in this chapter. 

(3)  https://echa.europa.eu/documents/10162/2324906/clp_introductory_en.pdf/b65a97b4-8ef7-4599-b122-

7575f6956027?t=1547546145023. 

(4)  https://eur-lex.europa.eu/eli/reg/2008/1272/oj. 

 
 
 
 
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Figure 1.  Schematic  representation  of  the  stepwise  approach  for  fulfilling  the  testing 
methodology for human health hazard for starting substances and constituents, for the purpose 
of the DWD 

Step 1
Does the relevant chemical species fall under any of the cases that justify the adoption of a limited acceptance methodology, 
as described in Section 1 of Annex VI to Commission Implementing Decision (EU) 2024/365?

Yes

No or limited, existing toxicological 
information is required.
Refer to Section 6.2 of DWD Guidance 
Volume II for deriving an appropriate 
MTCtap value

Ctap < 0.1 µg/l

Step 3
Is the relevant chemical 
species a monomer or other 
reactant of a main 
monomer?

No

Application for listing on the 
EUPL may not be necessary.
Refer to Section 2.1.4 of 
DWD Guidance Volume III

No

Step 2
Estimate migration and identify the 
relevant chemical species and their 
migration tier
(Chapter 5 of DWD Guidance Volume I)

Ctap ≥ 0.1 µg/l

Yes

Step 4
Collect all available information on 
toxicological properties of the relevant 
species 
(Chapter 3 of DWD Guidance Volume II)

Step 5
Evaluate all relevant available information
 (Chapters 3–4 of DWD Guidance Volume II)

Step 6
Generate any missing toxicological 
information to fulfil the information 
requirements in Annex V to Commission 
Implementing Decision (EU) 2024/365
(Chapter 6 of Guidance Volume I)

Step 7
Evaluate the new toxicological information 
(Chapters 3–4 of DWD Guidance Volume II)

Use available toxicological data to derive an 
appropriate MTCtap value for the relevant 
chemical species
(Chapter 6 of DWD Guidance Volume II)

NB: Ctap, concentration at the tap; MTCtap, maximum tolerable concentration at the tap. 

 
 
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Figure 2.  Schematic  representation  of  the  stepwise  approach  for  fulfilling  the  testing 
methodology for human health hazard for compositions, for the purpose of the DWD 

Step 1
Estimate migration according to Annex IV to Commission Implementing Decision (EU) 2024/365 to 
establish Ctap for the constituent or impurity present in the composition 
(Chapter 5. of DWD Guidance Volume I)

Step 2
Does the constituent/impurity have a MTCtap 
value in Annex V to Commission Decision 
Implementing Decision (EU) 2024/367?

YES

Step 3
Review the available toxicological 
information on the constituent/impurity to 
verify if the existing MTCtap value is still valid.
Is there new toxicological information?

NO

Step 4
Collect all available information on 
toxicological properties 
(Chapter 3 of DWD Guidance Volume II)

Step 5
Evaluate all relevant available information 
(Chapters 3–4 of DWD Guidance Volume II)

Step 5
Perform new testing if needed to fulfil the 
information requirements of 
Annex V to Commission Implementing 
Decision (EU) 2024/365
(Chapter 6 of DWD Guidance Volume I)

Step 6
Evaluate new information 
(Chapters 3–4 of DWD Guidance Volume II)

NO

No toxicity data 
required 

YES

Use available toxicological data to calculate 
MTCtap
(Chapter 6 of DWD Guidance Volume II)

Use existing MTCtap value shown in Annex V 
to Commission Implementing Decision (EU) 
2024/367

NB: Ctap, concentration at the tap; MTCtap, maximum tolerable concentration at the tap. 

 
 
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3.1.  Collection and evaluation of all available data 

3.1.1.  Collection of data (step 4 in Figure 1 and Figure 2) 

For  step 4  of  the  process,  various  sources  exist  for  gathering  all  available  information  on  the 
relevant chemical species. The eChemPortal ( 5) and the QSAR Toolbox ( 6) are recommended for 
the  collection  of  existing  information  on  toxicological  properties  and  for  the  determination  of 
potential application of non-test methods in the hazard assessment. Literature databases should 
also be considered. An additional list of sources to be considered during step 4 is available in 
Guidance  on  Information  Requirements  and  Chemical  Safety  Assessment –  Chapter R.3: 
Information gathering (7). 

3.1.2. 

Evaluation of the data (step 5 in Figure 1 and Figure 2) 

During  the  hazard  assessment,  it  is  very  important  to  evaluate  the  data  with  regard  to  their 
adequacy and completeness. This is particularly important for well-studied existing substances, 
for which there may be a number of test results available for each effect but some or all of these 
tests  may  not  have  been  carried  out  to  current  standards.  This  section  puts  forward  general 
guidelines on data evaluation. The term ‘adequacy’ is used here to cover the reliability of the 
available data and the relevance of those data for human hazard and risk assessment. In addition 
to the guidance provided in this section, Guidance on Information Requirements and Chemical 
Safety  Assessment –  Chapter R.4:  Evaluation  of  available  information (8),  provides  further 
guidance on assessing the relevance, reliability and adequacy of the information. 

3.1.2.1.  Completeness of data 

Section 2 of Annex V to Commission Implementing Decision (EU) 2024/365 gives the legal basis 
for toxicity data requirements for the relevant chemical species. Part 2 of Section 2 of Annex V 
to that decision specifies the general rules for the adaptation of the data requirements. 

3.1.2.2.  Adequacy of data 

The adequacy of data can be defined by two basic elements: 

• 

• 

reliability, covering the inherent quality of a test relating to test methodology and the way 
that the performance and results of the test are described; 

relevance, covering the extent to which a test is appropriate for a particular hazard or risk 
assessment. 

Reliable, relevant data  can be  considered valid for use in the  risk assessment. When there is 
more than one set of data for each effect, the greatest weight is attached to the most reliable 
and relevant. 

(5)  http://www.echemportal.org. 

(6)  http://www.qsartoolbox.org. 

(7)  https://echa.europa.eu/documents/10162/17235/information_requirements_r3_en.pdf/41895234-1125-4977-

b058-50a98e36fa48.   

(8)  https://echa.europa.eu/documents/10162/17235/information_requirements_r4_en.pdf/d6395ad2-1596-4708-

ba86-0136686d205e  

 
 
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The  evaluation  of  animal  test  data  with  respect  to  reliability 
is  outlined  below. 
Sections 3.1.2.2.3–3.1.2.2.5 consider issues specific to the reliability of human and in vitro data, 
relevance to humans and the (quantitative) structure–activity relationship ((Q)SAR). 

3.1.2.2.1. 

Reliability of data 

For relevant chemical species for which tests conducted in accordance with the EU test methods 
regulation  (Regulation  (EC)  No 440/2008)  and  in  compliance  with  the  principles  of  good 
laboratory practice (GLP) are available, many of the issues addressed in this section will not be 
relevant. 

For some substances, the test data available were generated prior to the requirements of GLP 
and the standardisation of testing methods. Those data may still be used for risk assessment, 
but the data and the methodology used must be evaluated in order to determine their reliability 
for assessment purposes. The evaluation needs expert judgement and must be transparent, so 
that the use made of a particular dataset is clearly justified. The requirements of the appropriate 
standardised test method and GLP principles should be regarded as a reference when evaluating 
the available test data. That is, studies carried out in accordance with current methods (e.g. the 
EU test methods regulation, Organisation for Economic Co-operation and Development (OECD) 
test guidelines programme (9) or United States Environmental Protection Agency (U.S. EPA) Test 
Guidelines ( 10),  appropriately  reported,  should  be  considered  the  most  reliable  for  risk 
assessment.  Klimisch  et  al.  (1997)  developed  the  following  scoring  system  to  assess  the 
reliability of data, particularly from toxicological studies. 

1 = Reliable without restrictions, which refers to ‘studies or data … generated according to 
generally  valid  and/or  internationally  accepted  testing  guidelines  (preferably  performed 
according to GLP) or in which the test parameters documented are based on a specific (national) 
testing  guideline …  or  in  which  all  parameters  described  are  closely  related/comparable  to  a 
guideline method’. 

2 = Reliable  with  restrictions,  which  refers  to  ‘studies  or  data …  (mostly  not  performed 
according  to  GLP),  in  which  the  test  parameters  documented  do  not  totally  comply  with  the 
specific  testing  guideline,  but  are  sufficient  to  accept  the  data  or  in  which  investigations  are 
described which cannot be subsumed under a testing guideline, but which are nevertheless well 
documented and scientifically acceptable’. 

3 = Not reliable, which refers to ‘studies or data … in which there are interferences between 
the measuring system and the test substance or in which organisms / test systems were used 
which are not relevant in relation to the exposure (e.g., unphysiological pathways of application) 
or  which  were  carried  out  or  generated  according  to  a  method  which  is  not  acceptable,  the 
documentation of which is not sufficient for an assessment and which is not convincing for an 
expert judgment’. 

4 = Not  assignable,  which  refers  to  studies  or  data  ‘ …  which  do  not  give  sufficient 
experimental details and which are only listed in short abstracts or secondary literature (books, 
reviews, etc.)’. 

The use of such scoring tools (e.g. the abovementioned Klimisch codes) allows the information 
to be ranked and organised for further review. This implies focusing on the most relevant tools, 
taking  into  account  the  hazard  being  measured  or  estimated.  The  evaluation  of  reliability  is 
performed  based  on  certain  formal  criteria,  using  international  standards  as  references.  The 

(9)  http://www.oecd.org/env/ehs/. 

(10)  https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/final-test-guidelines-pesticides-and-toxic.  

 
 
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scoring of information (e.g. using the Klimisch codes) should not exclude all unreliable data from 
further consideration by expert judgement because of the possible pertinence of these data in 
relation to the evaluated hazard. In general, some types of data that are not reliable (i.e. those 
for which documentation is not sufficient to make an assessment) and data for which it is not 
possible to assign reliability may be used only as supporting data. 

When looking at a test report, the assessor should consider the following. 

•  Whether the purity/impurities and the origin of the test material are reported. 

•  Whether a complete test report is available or the test has been described in sufficient detail, 
and whether the test procedure described is in accordance with the relevant guidelines and/or 
generally accepted scientific standards. If the information is considered to be reliable, it can 
be used for the hazard and risk assessment. 

•  Whether the reliability of the data cannot be fully established or the test procedure described 
differs  in  some  respects  from  the  test  guidelines  and/or  generally  accepted  scientific 
standards.  The  assessor  must  decide  in  those  cases  whether  the  data  will  be  taken  into 
consideration in the hazard and risk assessment and how they will be used (e.g. as supporting 
information where a reliable study has already been identified) or whether they should be 
regarded as invalid. 

•  Whether  the  following  factors,  among  others,  can  be  used  to  support  the  view  that  these 

data may be acceptable for use in a hazard and risk assessment: 

o 

there are other studies or calculations available on the relevant chemical species, and the 
data under consideration are consistent with them; 

o  other studies, for example on isomers with similar structure activity profiles, homologues, 
relevant precursors, breakdown products or other chemical analogues, are available, and 
the data under consideration are consistent with them; 

o  an  approximate  value  is  sufficient  for  taking  a  decision  on  the  result  of  the  risk 

characterisation. 

In principle, the same criteria apply to test data reported in the published literature. The 
amount of information presented will provide the basis for deciding on the reliability of the 
data reported. In general, publications in peer-reviewed journals are preferable. High-quality 
reviews may be used as supporting information. Summaries or abstract publications may also 
provide supporting material. 

3.1.2.2.2. 

Relevance of data 

In order to evaluate the relevance of the available data, it is necessary to judge, among other 
things, if an appropriate species has been studied, if the route of exposure is relevant for the 
population  and  if  the  substance  tested  is  representative  of  the  substance  as  supplied.  For 
relevant chemical species identified in the water intended for human consumption, the oral route 
is the only relevant route of exposure. 

Relevant human data of an adequate quality can sometimes be the best available data; however, 
more frequently, the available human, animal and non-animal data are considered together in 
order  to  reach  a  conclusion  about  the  relevance  to  humans  of  effects  observed  in  studies  in 
animals. 

 
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The evaluation of the relevance for humans of data from studies in animals is aided by use of 
data on the toxicokinetics, including metabolism of a substance in both humans and the animal 
species  used  in  the  toxicity  tests,  even  when  these  data  are  relatively  limited.  Clear,  well-
documented  evidence  of  a  species-specific  effect/response  (e.g.  light  hydrocarbon-induced 
nephropathy in the kidneys of male rats) should be used as justification for the conclusion that 
a particular effect is not expected to occur in humans exposed to the relevant chemical species. 

In  the  absence  of  such  information  (on  the  relevant  chemical  species  itself  or,  if  it  can  be 
scientifically  justified,  on  a  close  structural  analogue),  ‘threshold’  adverse  effects  observed  in 
studies in animals will normally be assumed to be likely to also occur in humans exposed to the 
relevant chemical species above a certain level of exposure. 

In any case, the dose–response relationships in the animal studies (or the severity of the effect, 
when  only  a  single  dose  was  tested)  are  also  assessed  as  a  part  of  the  hazard  and  risk 
assessment process. These assessments are taken into account at the risk characterisation stage 
when a judgement is made on the likelihood of occurrence of an adverse effect in humans at a 
particular level of exposure. 

Interpretation of the relevance of data derived from tests conducted in vitro should be taken into 
account whether the results seen have been observed or could be expected to occur (e.g. from 
knowledge of the toxicokinetics of the substance) in vivo. According to the validation procedures 
established  by  the  European  Centre  for  the  Validation  of  Alternative  Methods  (ECVAM),  the 
relevance of an alternative (non-animal) test, such as an in vitro test, is assessed according to 
the scientific basis of the test system (scientific relevance) and the predictive capacity (predictive 
relevance) of the prediction model, which is an algorithm for extrapolating from in vitro data to 
an in vivo end point (Worth and Balls, 2001). 

The  results  of  in  vitro  tests  (in  addition  to  the  standard  test  guideline  protocols  for  the 
assessment of specific end points, such as mutagenicity, that are mentioned in DWD Guidance 
Volume I)  may  provide  helpful  information,  which,  for  instance,  may  be  used  to  facilitate  the 
interpretation of the relevance for humans of data from studies in animals or to gain a better 
understanding of the mechanism of action of a substance. 

The  guidance  given  above  on  the  evaluation  of  the  adequacy  (relevance  and  reliability)  of 
information in general covers mainly the evaluation of data from animal studies. Some additional 
specific guidance is given below for human data, in vitro data and (Q)SARs. 

3.1.2.2.3. 

Human data 

The evaluation of human data usually requires a thorough critical assessment of the reliability 
of the data, considering some unique aspects distinct from the evaluation of animal data. 

Human data in the form of epidemiological studies or case reports can contribute to the hazard 
identification process as well as to the risk assessment process itself. 

Criteria for assessing the adequacy of epidemiology studies include an adequate research design, 
the  proper  selection  and  characterisation  of  the  exposed  and  control  groups,  adequate 
characterisation  of  exposure,  sufficient  length  of  follow-up  for  the  disease  as  an  effect  of  the 
exposure to develop, valid ascertainment of effect, proper consideration of bias and confounding 
factors, proper statistical analysis and a reasonable statistical power to detect an effect. These types 
of  criteria  have  been  described  in  more  detail  and  can  be  derived  from  epidemiology  textbooks 
(Hernberg, 1991; Checkoway et al., 2004; Rothman and Greenland, 2008). 

The results from human experimental studies, which are often not designed for the purpose of 
hazard  identification  and  hence  may  lack  sensitivity,  may  often  be  limited  by  several  factors, 

 
Guidance on the DWD: Volume II 
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such as a relatively small number of subjects, short duration of exposure and low dose levels, 
resulting in poor sensitivity in detecting effects. 

Therefore, epidemiological studies with negative results cannot prove the absence of an intrinsic 
hazardous property of a substance, but well-documented ‘negative’ studies of good quality may 
be  useful  in  the hazard  assessment.  Four  major  types  of  human  data  may  be  submitted:  (1) 
analytical  epidemiology  studies  on  exposed  populations,  (2)  descriptive  or  correlation 
epidemiology studies, (3) case reports and (4) in very rare, justified cases, controlled studies 
involving human volunteers. 

(1)  Analytical  epidemiology  studies  are  useful  for  identifying  a  relationship  between  human 
exposure  and  effects  such  as  biological  effect  markers,  early  signs  of  chronic  effects,  disease 
occurrence  and  increased  mortality  rates,  and  may  provide  the  best  data  for  hazard  and  risk 
assessment. Study designs include: 

• 

• 

• 

case–control (case–referent) studies, where groups of individuals with (cases) and without 
(controls/referents) a particular effect are identified and compared to determine differences 
in exposure; 

cohort studies, where groups of ‘exposed’ and ‘non-exposed’ individuals are identified and 
differences in effect occurrence are studied; 

cross-sectional studies, where a population (e.g. a workforce) is studied so that morbidity at 
a given point in time can be assessed in relation to concurrent exposure. 

The strength of the epidemiological evidence for specific health effects depends on, among other 
things, the type of analyses and the magnitude and specificity of the response. Confidence in 
the findings is increased when comparable results are obtained in several independent studies 
on populations exposed to the same agent under different conditions and using different study 
designs. 

Criteria  for  assessing  the  adequacy  of  epidemiology  studies  include  the  proper  selection  and 
characterisation  of  the  exposed  and  control  groups,  adequate  characterisation  of  exposure, 
sufficient  length  of  follow-up  for  disease  occurrence,  valid  ascertainment  of  effect,  proper 
consideration  of  bias  and  confounding  factors  and  a  reasonable  statistical  power  to  detect  an 
effect. 

(2)  Descriptive  epidemiology  studies  examine  differences  in  disease  rates  among  human 
populations  in  relation  to  age,  gender,  race  and  differences  in  temporal  or  environmental 
conditions. These studies are useful for identifying areas for further research but are not very 
useful for hazard and risk assessment. Typically, these studies can identify patterns or trends in 
disease  occurrence  over  time  or  in  different  geographical  locations  but  cannot  ascertain  the 
causal agent or degree of human exposure. 

(3) Case reports describe a particular effect in an individual or a group of individuals who were 
exposed to a substance. They may be particularly relevant when they demonstrate effects that 
cannot be observed in experimental animal studies. 

(4)  When  they  are  already  available,  well-conducted  controlled  human  exposure  studies  in 
volunteers, including low-exposure toxicokinetic (TK) studies,  can also be used in hazard and 
risk assessment in some rare cases. Those studies have to be conducted in line with the World 
Medical  Association  Declaration  of  Helsinki,  which  describes  the  general  ethical  principles  for 
medical research involving human subjects. 

 
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It should be emphasised that testing with human volunteers is strongly discouraged; however, 
when there are good quality data already available, they should be used as appropriate, in well-
justified cases. 

Experimental  human  toxicity  studies  must  not  be  conducted  specifically  for  the  purpose  of 
inclusion in an EUPL. 

3.1.2.2.4. 

In vitro data 

It can be expected that some of the available data will have been derived from studies conducted 
in vitro – the basic (and perhaps additional)  studies  on genotoxicity, for example. There may 
also  be  data  from  in  vitro  studies  on,  for  instance,  metabolism  and/or  mechanisms  of  action 
(including studies in cell cultures from different species), and various  aspects of toxicity (e.g. 
tests  for  cytotoxicity  in  different  types  of  cells,  macromolecule  binding  studies,  tests  using 
embryo culture systems, sperm motility tests). For any of these studies, their usefulness will be 
influenced by their adequacy in the light of some of the general criteria already discussed, for 
example how well the study is reported, how well the test material is characterised and to what 
extent the requirements of the method described in the EU test methods regulation (Regulation 
(EC) No 440/2008 ( 11)) and the principles of good in vitro method practice (12) have been met 
for the end point under consideration. 

However,  there  are  also  some  criteria  that  need  particular  attention  when  assessing  the 
adequacy of in vitro studies, for example: 

• 

• 

• 

• 

• 

• 

the range of exposure levels used, taking into account the toxicity of the substance towards 
the bacteria/cells, its solubility and, as appropriate, its effect on the pH and osmolality of the 
culture medium; 

the maintenance of effective concentrations of the volatile substances in the test system; 

the  use  of  an  appropriate  exogenous  metabolism  mix  (e.g.  S9  from  induced  rat  liver  or 
hamster liver) when necessary; 

the use of appropriate negative and positive controls as integral parts of the tests; 

the use of an adequate number of replicates (within the tests and of the tests); 

the use of the appropriate test system (e.g. appropriate cell lines). 

In  studies  conducted  in  accordance  with,  for  instance,  the  EU  test  methods  regulation,  OECD 
test guidelines programme (13) or US EPA test guidelines (14), the above criteria are evaluated 
against the specifications provided in the test guidelines. 

3.1.2.2.5. 

(Quantitative) structure–activity relationships 

When data do not exist for a given end point, or when data are limited, the use of structure–
activity  relationships  (SARs)  may  be  considered.  It  should  be  noted  that  (Q)SAR  models  are 

(11)  https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=celex%3A32008R0440. 

(12)  https://www.oecd.org/env/guidance-document-on-good-in-vitro-method-practices-givimp-9789264304796-

en.htm. 

(13)  http://www.oecd.org/env/ehs/. 

(14)  http://www.epa.gov/ocspp/pubs/frs/home/guidelin.htm. 

 
 
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currently not well developed for application in hazard and risk assessment, especially in relation 
to long-term mammalian toxicity. The (Q)SARs that are used for risk assessment purposes are 
usually of a more qualitative nature and do not address quantitative aspects. (Q)SARs may be 
of value in indicating a potential hazard, TK properties or the need for further testing. Additional 
guidance  is  provided  in  Guidance  on  Information  Requirements  and  Chemical  Safety 
Assessment – Chapter R.6: QSARs and grouping of chemicals (15). 

(Q)SAR predictions can be used together with other information in the weight of evidence (WoE) 
determination. When using (Q)SARs to predict a substance property, an assessment of both the 
model and the prediction is needed. A (Q)SAR model must be scientifically valid (using OECD 
principles (OECD, 2004, 2007)), and adequate and reliable documentation must be provided. A 
valid (Q)SAR model does not necessarily produce an acceptable prediction. For an acceptable 
(Q)SAR prediction, the input is correct, the substance falls within the applicability domain of the 
model, the prediction is reliable and the outcome is fit for the regulatory purpose. The validity 
of models and predictions can be assessed by using the OECD (Q)SAR assessment framework 
(OECD, 2023). 

Transparent documentation of the validity of the models must be provided as well as information 
relevant for judging the reliability of predictions for individual compounds or other comparable 
documentation. A (Q)SAR model  reporting format displays a description of the (Q)SAR model 
relative  to  the  five  OECD  (Q)SAR  validation  principles  in  a  systematic  and  summarised  way 
(OECD, 2004, 2007; minor update: OECD, 2023). The information about the (Q)SAR prediction 
is reported in the (Q)SAR prediction reporting format. An updated (Q)SAR prediction reporting 
format  template  was  published  in  2023,  and  it  reflects  the  newly  established  OECD  (Q)SAR 
prediction principles (OECD, 2023) ( 16). 

More information can be found in OECD (Q)SAR assessment framework documents (e.g. OECD, 
2023), in the ECHA guidance on (Q)SARs and grouping of chemicals (Chapter R.6 ( 17)) and in 
the ECHA practical guide How to Use and Report (Q)SARs (18). 

(Q)SAR models for predicting genotoxicity. In the context of the DWD, results from (Q)SAR 
models  that  met  all  conditions  described  under  Section 1.3  of  Annex XI  to  Regulation  (EC) 
No 1907/2006  on  the  registration,  evaluation,  authorisation  and  restriction  of  chemicals  (the 
REACH regulation) may be used as an alternative to experimental testing for genotoxicity, only 
for  relevant  chemical  species  constituents  or  a  non-intentionally  added  species,  if  it  can  be 
justified that the testing is technically not possible (e.g. it cannot be isolated and tested as such). 
In such cases, the recommendations described below have to be considered. 

There are hundreds of (Q)SAR models available in the literature for predicting genotoxicity test 
results (Honma, 2020). There are local (Q)SAR models, for relatively small sets of congeneric 
substances, that is, substances with the same basic structure and same mechanism of action, 
and global (Q)SAR models for a wide variety of non-congeneric substances. Global models may 
provide valuable predictive tools for estimating a number of mutagenic end points, if essential 
features of the information domain are clearly represented. However, the quality  of  reporting 
varies from model to model, and predictivity must be assessed case by case on the basis of clear 
documentation. 

(15)  http://echa.europa.eu/guidance-documents/guidance-on-information-requirements-and-chemical-safety-

assessment. 

(16)  https://one.oecd.org/document/ENV/CBC/MONO(2023)32/ANN2/en/pdf. 

(17)  https://echa.europa.eu/documents/10162/17224/information_requirements_r6_en.pdf. 

(18)  https://echa.europa.eu/documents/10162/17250/pg_report_qsars_en.pdf/407dff11-aa4a-4eef-a1ce-

9300f8460099.  

 
 
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A list of the available (free and commercial) predictive software for ecotoxicological, toxicological 
and  environmental  end  points,  including  mutagenicity  models,  has  been  compiled  within  the 
frame of an EU project (19). This website contains information on about 450 models, sorted by 
end  point.  The  Joint  Research  Centre  (JRC)  website  hosts  the  JRC  (Q)SAR  model  inventory, 
which is an inventory of information on (Q)SAR models that have been submitted to the JRC ( 20). 
This inventory currently contains a list of 154 models, of which 21 cover mutagenicity end points. 

The  bacterial  reverse  mutation  (Ames  test)  is  the  most  commonly  predicted  genotoxicity  end 
point for global models. The Ames test is reported to be well predicted, while the reliability of 
the  (Q)SAR  models  for  other  genotoxicity  assays /  end  points  is  still  quite  far  from  optimal 
(Benigni  et  al.,  2020).  However,  for  certain  classes  of  chemicals,  the  predictivity  of  other 
genotoxicity assays / end points can be quite  good, averaging the performance  for predicting 
the reverse mutation test (Ames test), while for other chemical classes the predictive statistics 
are poor, possibly also due to the poor availability of for these chemical classes. Recently, there 
has been significant progress in improving the predictive ability of (Q)SAR models, which have 
achieved  high  predictive  power,  including  for  predicting  clastogenicity  (Morita,  2019;  van 
Bossuyt, 2020). 

As a general trend, the combination of (Q)SAR models increases sensitivity, but at the expense 
of specificity. 

The  Danish  Environmental  Protection  Agency  and  the  (Q)SAR  group  at  the  National  Food 
Institute  at  the  Technical  University  of  Denmark  (DTU)  have  developed  the  Danish  (Q)SAR 
Database,  which  contains  predictions  from  a  number  of  mutagenicity  models.  In  addition  to 
assorted  reverse  mutation  test  (Ames  test)  models,  the  database  provides  predictions  of  the 
following  in  vitro  end  points:  chromosomal  aberrations  (CHO  and  CHL  cells),  mouse 
lymphoma/tk,  CHO/Hprt  gene  mutation  assays  and  unscheduled  DNA  synthesis  (rat 
hepatocytes). It also provides predictions of the following in vivo end points: Drosophila SLRL, 
mouse micronucleus, rodent dominant lethal, mouse sister chromatid exchanges in bone marrow 
and mouse comet assay. The Danish (Q)SAR Database is a repository of model predictions from 
more  than  200  (Q)SAR  models  and  is  considered  a  good  screening  tool.  All  organic  single-
constituent  substances  that  were  preregistered  or  registered  under  the  REACH  regulation 
(around 93 000) are included in the structure set. In addition, chemical structures from other 
relevant databases are included, leading to a structure set of more than 650 000 total / 600 000 
unique chemical structures. When possible, the end points have been modelled in three software 
systems,  Leadscope,  CASE  Ultra  and  SciQSAR.  Most  DTU-developed  models  and  a  number  of 
commercial models from MultiCASE have been modelled in two  or three  systems. For the set 
structure, predictions are provided in the different systems separately and as an overall battery 
prediction.  A  user  manual  with  information  on  the  individual  models,  including  training  set 
information and validation results, is available at the Danish (Q)SAR Database website. (Q)SAR 
model  reporting  format  documentation  of  all  DTU-developed  and  commercial  models  is  also 
available in the database. Predictions from a number of OECD QSAR Toolbox profilers have been 
included  as  supporting  information  to  the  (Q)SAR  predictions.  The  database  also  includes 
predictions  from  other  software  (e.g.  VEGA).  The  Danish  (Q)SAR  Database ( 21)  is  freely 
accessible and is also integrated into the OECD QSAR Toolbox. 

The  OECD  QSAR  Toolbox ( 22)  is  freely  available  software  developed  by  the  Laboratory  of 
Mathematical Chemistry, Burgas University, Bulgaria, driven by and in collaboration with ECHA, 

(19)  https://www.life-concertreach.eu/results/results-gateway/. 

(20)  https://jeodpp.jrc.ec.europa.eu/ftp/jrc-opendata/EURL-ECVAM/datasets/QSARDB/LATEST/qsardb.html. 

(21)  https://qsar.food.dtu.dk/. 

(22)  https://qsartoolbox.org/. 

 
 
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OECD  and  the  OECD  (Q)SAR  management  group.  It  is  an  expert  system  that  supports 
reproducible and transparent chemical hazard assessment. It offers functionalities for retrieving 
experimental data, simulating metabolism and profiling properties of chemicals. These pieces of 
information  and  tools  can  also  be  used  to  identify  structurally  and  mechanistically  defined 
analogues  and  chemical  categories,  which  can  serve  as  sources  for  read-across  and  trend 
analysis. 

Concerning mutagenicity, the OECD QSAR Toolbox covers information relevant for the bacterial 
reverse  mutation  test  (Ames  test),  in  vitro  chromosomal  aberration,  in  vivo  chromosomal 
aberration  (micronucleus  test)  and  genotoxic  carcinogenicity  end  points.  The  OECD  QSAR 
Toolbox includes a number of databases with relevant experimental data: REACH (ECHA), Food 
TOX  Hazard  (European  Food  Safety  Authority  (EFSA)),  bacterial  mutagenicity  (In  vitro 
Salmonella  typhimurium  mutagenicity  (Isssty)),  genotoxicity  and  carcinogenicity  (ECVAM), 
genotoxicity  (Outcome  and  Assessment  Information  Set  (OASIS)),  genotoxicity  pesticides 
(EFSA),  micronucleus  (In  vitro  micronucleus  test  (Issmic)),  micronucleus  (OASIS),  Toxicity 
Japan (Ministry of Health. Labour and Welfare (MHLW)) and the Transgenic Rodent Database. 
The OECD QSAR Toolbox allows the user to mine these databases for the purpose of (computer-
assisted) read-across analysis, looking for relevant structural analogues in these databases. It 
also offers profilers for predicting modes of actions that are relevant for mutagenicity, such as 
profilers  for  DNA  binding  by  OASIS  and  the  OECD  (Q)SAR  Toolbox,  DNA  alerts  for  Ames, 
chromosomal aberration and micronucleus test by OASIS, and in vitro and in vivo mutagenicity 
alerts  by  Intercom  Station  Software  (ISS).  Data  and  profilers  can  be  used  in  combination  to 
identify mechanistically and structurally relevant analogues for read-across predictions. Profilers 
identify structures associated with a hazard and are not predictive of the hazard or lack of hazard 
per se. There can be additional factors, such as metabolism, clearance and bioavailability, that 
may contribute to the genotoxicity potential of the substance. In addition, the profilers are often 
not  associated  with  defined  applicability  domains.  Unbiased  subcategorisation  is  extremely 
important when using the profilers in the OECD QSAR Toolbox. 

In  addition  to  the  data  and  profilers,  the  OECD  QSAR  Toolbox  includes  predictions  of 
mutagenicity from a number of (Q)SAR models from the Danish (Q)SAR Database. 

The Danish (Q)SAR Database and the OECD QSAR Toolbox profilers can support predictions for 
multiple  genotoxicity  end  points  and  may  assist  those  making  WoE  decisions  regarding  the 
mutagenic potential of untested substances. 

For mutagenicity predictions, the potential of the substances to generate metabolites of concern 
should  also  be  considered.  Some  models  for  predicting  genetic  toxicity  include  a  metabolic 
simulator  and  prediction  of  metabolites  for  genotoxicity  potential.  For  models  not  including  a 
metabolic  simulator,  separate  in  silico  approaches  to  predict  the  likely  metabolites  based  on 
molecular structure are available. Reliability of simulated metabolism is discussed in Dermen et 
al. (2022). In some cases, models may implicitly assume metabolism, such as in the use of the 
aromatic amine alert (OECD QSAR Toolbox, Derek Nexus, VEGA, MultiCASE) as an indication of 
mutagenic potential. Metabolism can also explain some differences between in vitro and in vivo 
genotoxicity (Petkov et al., 2022). In addition, a new (Q)SAR modelling concept was introduced 
in  which  the  potency  is  related  to  the  amount  of  DNA  adducts  as  an  element  of  metabolism 
prediction. 

When using (Q)SARs to predict a relevant chemical species property, an assessment of both the 
model and the prediction is needed. Further guidance on (Q)SARs can be found in Section R.6.2 
of  Guidance  on  Information  Requirements  and  Chemical  Safety  Assessment –  Chapter R.6: 
QSARs  and  grouping  of  chemicals  and  in  the  OECD  (Q)SAR  assessment  framework,  which 
provides  guidance  and  practical  advice  on  how  to  assess  the  validity  of  models  and  their 

 
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predictions  (OECD,  2023) (23).  The  assessment  framework  states  that  the  validity  of  a  model 
should be assessed according to the OECD validation principles for (Q)SARs (OECD, 2004, 2007), 
while the validity of predictions can be assessed against the newly established principles for the 
assessment  of  (Q)SAR  predictions  and  results  presented  in  the  assessment  framework 
guidance ( 24). 

For prediction of gene mutation in bacteria, it is important that all relevant strains of the Ames 
test  are  addressed.  Metabolic  activation  should  be  taken  into  account  for  adequacy  and 
equivalence  to  tests  (in  order  to  make  the  predictions  suitable  for  meeting  the  information 
requirements of the DWD). For statistical models, verification that the substance falls within the 
applicability  domain  and  information  on  analogues  supporting  the  predictions  are  important. 
Negative  predictions  from  an  alert-based  system  such  as  Derek  Nexus,  for  example,  can  be 
considered only in the vicinity of very similar compounds that tested negative in the respective 
tests,  assuming  that  all  strains  and  metabolic  activation  are  covered.  These  can  be  searched 
outside  Derek  Nexus  (e.g.  using  Sarah  Nexus,  Vitic  or  another  database  source,  such  as  the 
OECD QSAR Toolbox ( 25)). The documentation that Derek Nexus provides in the results window, 
however, is currently not sufficient to assess a prediction. 

The  Danish  (Q)SAR  Database  includes  statistical  models  from  SciQSAR,  Leadscope  and  CASE 
Ultra models for the Ames test. However, the documentation for the Danish (Q)SAR Database 
does not always allow users to verify that the substance falls within the applicability domain and 
verify  information  from  analogue  substances  to  support  the  prediction.  Information  on  the 
models is available from the website ( 26). 

If well-documented and applicable (Q)SAR data are available, they should be used to help reach 
the decision points described below in this section. In many cases, the accuracy of such methods 
will be sufficient to enable either a test or a specific regulatory decision to be made. In other 
cases, the uncertainty may be considered too high, and further data will be needed. 

The accuracy of available methods may be assessed using substances that were not originally 
included  in  the  training  set  of  the  models  (so-called  external  validation).  There  was  an 
international challenge project for predicting the bacterial reverse mutation test (Ames test), the 
results of which are described in Honma et al. (2019). Evaluating new data on mutagenicity can 
lead to expansion of the training sets (Amberg et al., 2019; Petkov et al., 2019a). 

Documentation can include reference to a related and relevant substance or group of substances 
that leads to the conclusion of concern or lack of concern. This can be presented in accordance 
with a scientific logic (read-across justification) or sometimes as a mathematical relationship of 
chemical similarity. It should be noted that when an in silico tool such as the OECD QSAR Toolbox 
is  used  as  a  first  step,  to  find  analogues  for  performing  read-across  of  data  but  not  to  make 
predictions  based  on  (Q)SAR,  this  is  considered  a  read-across  approach  and,  as  such,  the 
justification and predictions should comply with the conditions of a read-across adaptation (see 
Section 3.1.6). 

The lack of mechanistic justification may, depending on the application context, limit the use of 
(Q)SAR predictions, particularly for regulatory decisions. Workflows based on a combination of 
mechanistic (Q)SAR, read-across analysis and expert knowledge may be derived to allow users 
to make a transparent decision on the final prediction based on WoE (Petkov et al., 2019b). In 
the  case  of  consistent  predictions,  expert  input  may  not  be  needed  to  make  a  final  decision. 

(23)  https://one.oecd.org/document/ENV/CBC/MONO(2023)32/en/pdf.  

(24)  https://ec.europa.eu/eurostat/documents/64157/4392716/ESS-QAF-V2.0-final.pdf. 

(25)  https://qsartoolbox.org/. 

(26)  https://qsar.food.dtu.dk/. 

 
 
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Nonetheless,  expert  input  may  be  useful  to  expand  the  set  of  read-across  analogues  from 
literature  sources  and/or  to  provide  a  rationale  for  the  end-point-specific  similarity  between 
source analogue(s) and the target substance and, not least, to assess how the differences in the 
chemical structure between these may affect the end point under assessment. Advice on how to 
interpret some freely available models is provided by Mombelli et al. (2016). The WoE approach 
for Ames test predictions is also discussed in Mombelli et al. (2022). 

Substances for which no test data exist or for which testing is technically not possible represent 
a special case in which reliance on non-testing data may be absolute. Many factors will dictate 
the acceptability of non-testing methods in reaching a conclusion based on no tests at all. It may 
be  discussed  whether  WoE  decisions  based  on  multiple  genotoxicity  and  carcinogenicity 
estimates can equal or exceed those obtained by one or two in vitro tests, and whether general 
rules  for  adaptation  of  the  standard  testing  regime  as  described  in  Part 2  of  Annex V  to 
Commission Implementing Decision (EU) 2024/365 may be invoked based on such estimates. 
This must be considered on a case-by-case basis. 

(Q)SAR  models  are  continuously  updated  to  improve  predictions,  with  new  versions  typically 
released  on  a  yearly  basis.  It  is  important  to  understand  the  impact  of  model  updates  on 
mutagenicity predictions over time. Such analysis has been done, for instance, by Hasselgren et 
al. (2020) on computational methods used for the prediction of the mutagenic properties of drug 
impurities. 

(Q)SAR models for predicting repeated dose toxicity (including target organ toxicity, 
toxicity to reproduction and development). Overall, (Q)SAR approaches are currently not 
well validated for  repeated dose toxicity and, consequently, no firm  recommendations  can be 
made concerning their routine use in a testing strategy in this area. One of the reasons is that 
there are a large number of potential targets/mechanisms associated with repeated dose toxicity 
that  currently  cannot  be  adequately  covered  by  a  battery  of  (Q)SAR  models.  Therefore,  a 
negative  result  from  current  (Q)SAR  models  without  other  supporting  evidence  cannot  be 
interpreted as demonstrating a lack of a toxicological hazard or a need for hazard classification. 
Another  limitation  of  (Q)SAR  modelling  is  that  dose–response  information,  including  the 
N(L)OAEL, is not provided. Similarly, a valid (Q)SAR model may identify a potential toxicological 
hazard, but, because of limited confidence in this approach, such a result would not be adequate 
to support hazard classification. 

It  has  to  be  acknowledged,  however,  that  there  are  emerging  models,  some  of  which  are 
currently under development and have not undergone the OECD validation process yet. 

In  some  cases,  (Q)SAR  models  could  be  used  as  part  of  a  WoE  approach,  when  considered 
alongside other data, provided the input is correct, the substance falls within the applicability 
domain of the model, the prediction is reliable and the outcome is fit for the regulatory purpose. 
The validity of models and predictions can be assessed by using the OECD (Q)SAR assessment 
framework  (OECD,  2023).  In  addition,  (Q)SARs  can  be  used  as  supporting  evidence  when 
assessing  the  toxicological  properties  by  read-across  within  a  substance  grouping  approach, 
providing the applicability domain is appropriate. Positive and negative (Q)SAR modelling results 
can be of value in a read-across assessment and for classification purposes. 

3.1.3.  Read-across approach 

In the context of the DWD, read-across is provided as an alternative to testing to predict the 
toxicological properties of relevant chemical species. 

If  the  read-across  approach  is  used,  the  conditions  stated  in  Section 1.5  (on  the  read-across 
approach) of Annex XI to Regulation (EC) No 1907/2006 should be met. 

 
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The read-across approach needs to be adequately and appropriately documented to support the 
read-across hypothesis and predictions. 

The  read-across  assessment  framework  (RAAF)  documents  of  March  2017 (27) (28)  describe  a 
general framework and principles for the scientific assessment of the suitability of a read-across 
approach  based  on  different  scenarios.  These  scenarios  are  selected  according  to  the  type  of 
read-across  approach  used  (analogue  or  category  approach),  the  basis  for  the  read-across 
hypothesis  ((bio)transformation  of  the  analogues  into  common  compound(s)  or  different 
compounds  having  qualitatively  similar  properties)  and  whether  quantitative  variations  in  the 
predicted properties are expected between the different analogues. 

To  justify  the  validity  of  a  read-across  approach,  one  important  element  of  the  read-across 
assessment  framework  is  the  need  to  specify  why  the  commonalities  between  two  or  more 
analogue structures suggest a similar biological action. A justification also needs to be provided 
as to why structural dissimilarities are not expected to result in dissimilar biological actions. 

To  assess  the  suitability  of  selected  analogues  as  source  substances,  the  following  general 
questions also need to be addressed. 

• 

Is the same end point considered? 

•  Are there any additional functional groups or additional substituents that may influence the 

reactivity and mutagenicity potential (applicability domain considerations)? 

•  Are the physico-chemical parameters similar (applicability domain considerations)? 

•  Are there impurities that influence the toxicity profile? 

• 

Is the likely chemical mechanism the same? 

A read-across approach can also support a conclusion for a property within a WoE approach. 

3.1.4.  Weight of evidence (WoE) 

When applying the WoE approach, the conditions stated in Section 1.2 of Annex XI to Regulation 
(EC)  No 1907/2006  (the  REACH  regulation)  should  be  met.  In  addition,  the  provisions  of 
Section 1.1.1  of  Annex I  to  Regulation  (EC)  No 1272/2008  (the  CLP  regulation)  should  be 
considered. 

The  REACH  regulation,  Annex XI,  Section 1.2,  states  that  there  may  be  sufficient  WoE  from 
several  independent  sources  of  information  to  enable,  through  a  reasoned  justification,  a 
conclusion on the information requirement, while the information from each single source alone 
is insufficient to fulfil the information requirement. 

According to Section 1.1.1 of Annex I to the CLP regulation, ‘weight of evidence determination 
means  that  all  available  information  bearing  on  the  determination  of  hazard  is  considered 
together, such as the results of suitable in vitro tests, relevant animal data, information from 
the  application  of  the  category  approach  (grouping,  read-across),  (Q)SAR  results,  human 

(27)  https://echa.europa.eu/support/registration/how-to-avoid-unnecessary-testing-on-animals/grouping-of-

substances-and-read-across. 

(28)  https://echa.europa.eu/documents/10162/13630/raaf_uvcb_report_en.pdf/3f79684d-07a5-e439-16c3-

d2c8da96a316. 

 
 
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experience  such  as  occupational  data  and  data  from  accident  databases,  epidemiological  and 
clinical studies and well-documented case reports and observations. The quality and consistency 
of the data shall be given appropriate weight. Information on substances or mixtures related to 
the substance or mixture being classified shall be considered as appropriate, as well as site of 
action and mechanism or mode of action study results. Both positive and negative results shall 
be assembled together in a single weight of evidence determination.’ 

Relevant chemical species for which no test data exist, for example relevant chemical species 
that  cannot  be  tested,  present  a  special  case  in  which  reliance  on  non-testing  data  may  be 
absolute.  Many  factors  will  dictate  the  acceptability  of  non-testing  methods  in  reaching  a 
conclusion based on no tests at all. It has yet to be established whether WoE decisions based on 
multiple genotoxicity and carcinogenicity estimates can equal or exceed those obtained by one 
or two in vitro tests. This must be considered on a case-by-case basis. 

WoE  is  based  on  expert  judgement.  This  expert  judgement  has  to  be  made  transparent  and 
understandable  by  documenting  all  information  used,  all  steps  carried  out  in  the  evaluation 
process and all conclusions drawn. Furthermore, the recommendations on how to perform WoE 
given  in  EFSA’s  guidance  on  the  use  of  the  WoE  approach ( 29)  and  in  the  ECHA  guidance 
(Chapter R.4) ( 30) can be followed. 

3.2.  Toxicokinetics 

The aim of this section is to provide a general overview of the main principles of toxicokinetics 
and  guidance  on  the  use  of  TK  information  in  the  human  health  risk  assessment  of  relevant 
chemical species. For the generation of new TK data, this guidance document should be used 
together with DWD Guidance Volume I. 

3.2.1.  Definitions and uses of toxicokinetic studies 

TK studies evaluate the fate of a substance in an organism, that is, its absorption, distribution, 
metabolism  and  excretion  (ADME)  (see  ECETOC,  2006;  Regulation  (EC)  No 440/2008 
(Section B.36); and OECD Test Guideline (TG) 417 (2010)). 

Toxicokinetics may help in understanding the results from both in vivo and in vitro toxicology 
studies by  elucidating the ADME of the test  substance. Basic  TK parameters determined from 
these studies will also provide information on the potential for accumulation of the test substance 
in  tissues  and/or  organs  and  the  potential  for  induction  or  inhibition  of  biotransformation  by 
measuring the circulating moieties (parent substance and its metabolites). 

Adequate  TK  data  can  further  be  used  for  in  silico  predictions  and  physiologically  based 
TK/pharmacokinetic modelling or for grouping of substances and read-across approaches. 

The TK data should, however, not be used for dose selection or to disregard positive results for 
hazard evaluation in the absence of remarkable and unspecific general toxicity. 

(29)  https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2017.4971. 

(30)  https://echa.europa.eu/documents/10162/17235/information_requirements_r4_en.pdf/d6395ad2-1596-4708-

ba86-0136686d205e?t=1323782558175. 

 
 
 
 
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3.2.2.  Main principles of toxicokinetic studies 

TK  studies  are  designed  to  obtain  species-  and  route-dependent  parameter  data  on  the 
concentration–time  course  of  a  parent  substance  and  its  metabolites  in,  for  instance,  blood, 
urine, bile, faeces, exhaled air and organs. 

According to OECD TG 417 on TK studies, data on the following parameters should be assessed: 

•  half-life and potential for accumulation under repeated or continuous exposure; 

• 

rate and extent of absorption and bioavailability; 

•  distribution of the relevant chemical species in the body; 

•  biotransformation; 

• 

• 

• 

rate and extent of pre-systemic (first-pass) and systemic metabolism after oral or inhalation 
(if available) exposure; 

rate  and  extent  of  excretion  in  the  urine  or  faeces,  via  exhalation,  and  in  other  biological 
fluids (e.g. milk, bile, sweat, etc.); 

information on enterohepatic circulation. 

Enterohepatic  circulation  may  pose  problems  for  route-to-route  extrapolation  since  systemic 
availability after oral administration may be greater than after non-oral administration. This will 
result in an area under the curve (AUC) (which reflects both the absorption / systemic availability 
of  the  test  substance  and  the  extent  of  recirculation).  As  the  relative  extent  of  absorption 
following  different  routes  of  exposure  is  often  calculated  from  the  ratio  of  AUCs  by  different 
routes,  the  absorption  after  oral  exposure  may  be  overestimated  when  enterohepatic 
recirculation  takes  place.  For  this  reason,  TK  studies  are  preferably  conducted  using  the  oral 
route  to  administer  the  substance.  Integrating  TK  data  into  toxicity  studies  can  improve  the 
quality of  risk assessment in many  ways, including aiding understanding of the differences  in 
responses or sensitivity between individual animals, species or life stages. 

In the absence of in vivo data, standardised in vitro / ex vivo assays may, to a limited extent, 
provide data on some of the TK parameters. These include parameters of metabolic steps, such 
as  Vmax (31),  Km (32)  and  intrinsic  metabolic  clearance,  permeation  rates  (e.g.  intestinal, 
placental,  blood–brain  barrier ( 33))  and  the  distribution  coefficient.  Physiologically  based  TK 
modelling techniques may be used to simulate the concentration–time profile in blood and at the 
target site. 

However,  these  assays  are  non-stand-alone  methods  that  can  be  used  prior  to  an  in  vivo  TK 
study. These include: 

(31)  Maximum velocity; reflects how fast the enzyme can catalyse the reaction. 

(32)  Michaelis constant; describes the substrate concentration at which half the enzyme’s active sites are occupied by 

the substrate. 

(33)  Currently,  there  is  no  standardised  and  regulatory-process-compliant  in  vitro  methods  for  permeation  rate  via 

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•  protein binding and binding to erythrocytes, if relevant (in vitro / ex vivo studies); 

•  hepatic  metabolic  clearance  with  primary  human  hepatocytes  or  liver  cell  lines  (in  vitro 

studies); 

• 

intestinal permeability (the in vitro Caco-2 permeability assay). 

3.2.2.1.  Accumulation 

Everything in a biological system has a biological half-life, that is, a measure of how long it will 
stay  in  that  system  until  it  is  lost  by  mainly  excretion,  degradation  or  metabolism.  To  put  it 
another way, the amount of a substance eliminated from the blood in a unit of time is the product 
of clearance (the volume of blood cleared per unit of time) and concentration (the amount of a 
compound per unit of volume). For the first-order reactions, clearance is a constant value that 
is  a  characteristic  of  a  substance.  If  the  absorption  rate  of  a  substance  into  an  organism  is 
greater  than  the  rate  at  which  the  substance  is  eliminated,  the  organism  is  said  to  be 
accumulating the substance. When the internal concentration has increased so that the amount 
eliminated equals the amount of substance absorbed, there will be a constant concentration, a 
steady state. The extent of accumulation reflects the relationship between the body burden and 
the  steady  state.  Species  differences  in  clearance  will  determine  the  difference  in  the  steady 
state and body burden between experimental animals and humans (Kroes et al., 2004). 

3.2.3.  Prediction of toxicokinetics 

3.2.3.1.  Absorption 

Absorption is a function of the potential for a substance to cross biological membranes. These 
membranes  consist  of  lipidic  layers  and  aqueous  phases,  which  require  the  substance  to  be 
soluble both in lipid and in water in order to cross through the membrane by passive diffusion. 
Therefore, apart from molecular weight, the partition coefficient (log P) – or the n-octanol–water 
partition  coefficient  (log  Kow) –  value  and  the  water  solubility  of  a  substance  are  useful 
parameters  in  understanding  the  biological  mechanisms  behind  absorption.  The log  Kow  value 
provides information on the relative solubility of the substance in water and in the hydrophobic 
solvent  octanol  (used  as  a  surrogate  for  lipids).  A  log  Kow  value  of  > 0  indicates  that  the 
substance is lipophilic and, therefore, more soluble in octanol than in water. A log Kow value of 
< 0 indicates that the substance is hydrophilic and, hence, more soluble in water than in octanol. 
In  general,  log  Kow  values  between  – 1  and  4  are  favourable  for  absorption.  Nevertheless,  a 
substance  with  such  a  log  Kow  value  can  be  poorly  soluble  in  lipids  and,  hence,  not  readily 
absorbed  when  its  water  solubility  is  very  low.  It  is  therefore  important  to  consider  both  the 
water solubility of a substance and its log Kow value when assessing the absorption potential of 
a  substance.  Substances  that  do  not  absorb  by  passive  diffusion  can  cross  the  membrane  by 
facilitated diffusion, active transport or pinocytosis. 

Once absorbed, the substance enters the blood system for transportation to the liver, where the 
substance may undergo first-pass metabolism. The parent compound and/or its metabolites may 
then  continue  through  the  circulatory  systemic  and  further  excreted  via  urine  or  into  the  bile 
(known  as  the  first-pass  effect  or  pre-systemic  elimination).  Large  molecules  or  lipophilic 
substances,  on  the  other  hand,  may  instead  enter  the  lymphatic  system.  Substances  in  the 
lymphatic fluid are then emptied into the subclavian veins and, thereby, will bypass the liver and 
its first-pass metabolism. 

Oral/gastrointestinal  (GI)  absorption.  The  GI  tract  is  an  organ  with  a  large  absorptive 
surface area that stretches from the mouth to the rectum. Absorption of a substance can occur 
at different sites along the GI tract and with different transport mechanisms. The small intestine 

 
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has a very large surface area, and the transit time through this section is the longest, making 
this  the  predominant  site  of  absorption  within  the  GI  tract.  Compared  with  that  of  the  small 
intestine, the rate and extent of absorption within the large intestine is low. Table 1 provides an 
overview of different types of data that can be considered for estimating GI absorption, including 
physico-chemical characteristics of a substance, type of dosing vehicle used in the in vivo study 
and the possibility of the hydrolysis of a substance. Regarding the hydrolysis possibility, it should 
also  be  noted  that  substances  could  undergo  chemical  changes  in  the  GI  fluids  as  a  result  of 
metabolism  by  intestinal  cells,  GI  flora  and/or  enzymes  released  into  the  GI  tract,  or  by 
spontaneous  hydrolysis.  These  changes  will  alter  the  physico-chemical  characteristics  of  the 
substance; hence, predictions based on the properties of the parent substance may no longer 
apply (see Appendix 1-1 to Guidance on the Biocidal Products Regulation – Volume III human 
health –  Assessment  &  evaluation  (Parts B+C) (34)  for  a  detailed  list  of  physiological  factors, 
data on stomach and intestine pH, and data on transit time in the intestine, among other things). 
In addition, the presence of food in the GI tract can affect (generally decrease) the GI absorption 
considerably.  In  that  sense,  absorption  can  differ  considerably  between  routes  of  oral 
administration, for example via gavage or diet. 

For ionic substances (i.e. acids and bases), one consideration that could influence the absorption 
is the varying pH of the GI tract. It is generally thought that ionised substances do not readily 
diffuse across biological membranes. Therefore, when assessing the absorption potential of an 
acid or a base, knowledge of its pKa (the pH at which 50 % of the substance is in ionised form 
and 50 % in non-ionised form) is advantageous. Absorption of acids is favoured at pH < pKa, 
whereas absorption of bases is favoured at pH > pKa. 

Other mechanisms by which substances can be absorbed in the GI tract include the passage of 
small  water-soluble  molecules  with  a  molecular  weight  up  to  around  200 Da.  These  small 
molecules can pass through the aqueous pores or be carried through with the bulk passage of 
water. 

The absorption of highly lipophilic substances (log Kow ≥ 4), on the other hand, may be limited 
by  the  inability  of  such  substances  to  dissolve  into  the  GI  fluids  and  make  contact  with  the 
mucosal  surface.  However,  the  bile  salts  in  the  GI  tract  aid  in  decomposing  highly  lipophilic 
substances  into  simpler  molecules,  which  are  then  formed  into  micelles  that  enhance  the 
absorption of such substances (Watkins and Klaassen, 2018). Substances absorbed as micelles 
enter the circulation via the lymphatic system, thereby bypassing the liver. Although particles 
and large molecules with a molecular weight of around 1000 g/mol (or Da) would normally be 
considered too large to cross biological membranes, small amounts of such substances may be 
transported into epithelial cells by pinocytosis or persorption. 

(34)

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Table 1. How different types of data can be used to interpret oral/GI absorption 

Type of data 

What they tell us  

Structure 

It  may  be  possible  to  identify  ionisable  groups  within  the  structure  of  the 
molecule. Groups containing oxygen, sulphur or nitrogen atoms are potentially 
ionisable (e.g. thiol (SH), sulphonate (SO3H), hydroxyl (OH–), carboxyl (COOH) 
or amine (NH2)). 

Molecular weight 

Generally,  the  smaller  the  molecule  the  more  easily  it  may  be  taken  up. 
Molecular  weights  of  < 500 g/mol  are 
for  absorption;  
molecular weights of > 1 000 g/mol do not favour absorption. 

favourable 

Particle size 

Water solubility 

Log Kow 

Dosing vehicle 

Oral toxicity data 

Hydrolysis test 

Generally,  solids  have  to  dissolve  before  they  can  be  absorbed.  It  may  be 
possible  for  particles  in  the  nanometre  size  range  to  be  taken  up  through 
pinocytosis or passive transport (Amornwachirabodee et al., 2018; Manzanares 
and Ceña, 2020). The absorption of a substance that was administered as a dry 
powder  (e.g.  as  large  particles,  several  hundreds  of  micrometres  in  diameter, 
mixed in the diet) or in a suspension may be reduced because of the time taken 
for the particles to dissolve. This would be particularly relevant for substances 
with low solubility in water. 

Water-soluble substances will readily dissolve in the GI fluids. Absorption of very 
hydrophilic substances via passive diffusion may be limited by the rate at which 
the substance partitions out of the GI fluids. However, if the molecular weight is 
low (< 200 g/mol), the substance may pass through aqueous pores or be carried 
through the epithelial barrier by the bulk passage of water. 

Moderate log Kow values (between – 1 and 4) are favourable for absorption by 
passive  diffusion.  Any  lipophilic  compound  may  be  taken  up  by  micellular 
solubilisation,  but  this  mechanism  may  be  of  particular  importance  for  highly 
lipophilic compounds (log Kow > 4), particularly those that have low solubility in 
water (≤ 1 mg/l) and would otherwise be poorly absorbed. 

If  the  substance  has  been  dosed  using  a  vehicle,  the  water  solubility  of  the 
vehicle  and  the  vehicle/water  partition  coefficient  of  the  substance  may  affect 
the  rate  of  uptake.  Compounds  delivered  in  aqueous  media  are  likely  to  be 
absorbed more rapidly than those delivered in oils. Compounds delivered in oils 
that can be emulsified and digested, such as corn oil or arachis oil, are likely to 
be absorbed to a greater degree than those delivered in non-digestible mineral 
oil (liquid petrolatum). 

If  signs  of  systemic  toxicity  are  present,  then  absorption  has  occurred (a). 
Coloured urine and/or internal organs can also provide evidence that a coloured 
substance has been absorbed. In addition, some clinical signs such as a hunched 
posture could be due to discomfort caused by irritation or simply the presence 
of  a  large  volume  of  test  substance  in  the  stomach,  and  reduced  feed  intake 
could be due to an unpalatable test substance. It must therefore be clear that 
the effects that are being cited as evidence of systemic absorption are genuinely 
due to absorbed test substance and not to local effects at the site of contact. 

The hydrolysis test (EU testing method C.7; OECD TG 111) provides information 
on the half-life of the substance in water at 50 °C and pH values of 4.0, 7.0 and 
9.0.  The  test  is  conducted  using  a  low  concentration,  0.01 M  or  half  the 
concentration  of  a  saturated  aqueous  solution  (whichever  is  lower).  Since  the 
temperature at which this test is conducted is much higher than that in the GI 

 
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Type of data 

What they tell us  

tract, this test will not provide an estimate of the actual hydrolysis half-life of 
the substance in the GI tract. However, it may indicate that the parent compound 
is present in the GI tract for only a limited period of time. Hence, TK predictions 
based on the characteristics of an easily hydrolysable parent compound may be 
of limited relevance. 

(a) 

Ensure that systemic effects do not occur secondary to local effects. 

Respiratory  and  dermal  absorption.  Inhalation  and  dermal  exposure  are  not  the  most 
relevant routes of exposure when it comes to relevant chemical species identified in the water 
intended for human consumption. Therefore, if an inhalation or dermal study is provided to meet 
the  information  requirements  under  the  DWD,  the  applicant  needs  to  provide  a  scientifically 
robust justification for choosing a route that is different from the oral route of administration. 
Nevertheless,  as  it  cannot  be  excluded  that  humans  will  be  exposed  dermally  and/or  by 
inhalation  to  drinking  water,  positive  results  seen  after  exposure  via  these  routes  cannot  be 
discarded. Furthermore, if such a study is used for hazard characterisation, extrapolation from 
respiratory/dermal  to  oral  absorption  has  to  be  done.  If  this  is  the  case,  the  applicants  are 
advised to consult Guidance on the Biocidal Products Regulation – Volume III human health – 
Assessment & evaluation (Parts B+C), Tables 2 and 3 (35). 

3.2.3.2.  Distribution 

The concentration of a substance in blood or plasma (blood level) is dependent on the dose, on 
the rate of absorption, distribution and elimination, and on the affinity to a tissue. Tissue affinity 
is  usually  described  using  a  parameter  known  as  the  volume  of  distribution,  which  is  a 
proportionality factor between the amount of substance present in the body and the measured 
plasma or blood concentration. The larger the volume of distribution, the lower the blood level 
will be for a given amount of substance in the body. A particularly useful term is the volume of 
distribution at a steady state (Vdss). At a steady state, all distribution phenomena are completed, 
the various compartments of the body are in equilibrium, and the rate of elimination is exactly 
compensated by the rate of absorption. In non-steady-state situations, the distribution volume 
varies with time except in the simplest case of a single-compartment model (this model assumes 
that the entire body acts as a single, uniform compartment and the substance can enter or leave 
the body, that is, the model is ‘open’). In theory, a steady state can be physically reached only 
in  the  case  of  a  constant  zero-order  input  rate  and  at  stable  first-order  distribution  and 
elimination  rates.  However,  many  real  situations  are  reasonably  close  to  a  steady  state,  and 
reasoning at a steady state is a useful method in kinetics. 

The rate at which highly water-soluble molecules distribute may be limited by the rate at which 
they cross cell membranes, and the access of such substances to the central nervous system 
(CNS)  or  testes  is  likely  to  be  restricted  by  the  blood–brain  and  blood–testes  barriers  (Slitt, 
2018).  It  is  not  clear  what  barrier  properties  the  placenta  may  have.  However,  species 
differences in transplacental transfer may occur due to varying placental structure and metabolic 
capacity of the placenta and placental transporters. 

Although protein binding can limit the amount of a substance available for distribution, it will 
generally  not  be  possible  to  determine  from  the  available  data  which  substances  will  bind  to 
proteins and how strongly they will bind. Furthermore, if a substance undergoes extensive first-

(35)

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pass metabolism, predictions made on the basis of the physico-chemical characteristics of the 
parent substance may not be applicable. 

Table 2  provides  an  overview  of  data  that  can  be  considered  for  estimating  substance 
distribution. 

Table 2. How different types of data can be used to interpret substance distribution  

Type of data 

What they tell us  

Molecular weight 

In general, the smaller the molecule, the wider the distribution. 

Water solubility 

Log Kow 

Target organs 

Signs of toxicity 

Trace elements 

Small  water-soluble  molecules  and  ions  will  diffuse  through  aqueous  channels 
and  pores.  The  rate  at  which  very  hydrophilic  molecules  diffuse  across 
membranes could limit their distribution. 

If the molecule is lipophilic (log Kow > 0), it is likely to distribute into cells, and 
the  intracellular  concentration  may  be  higher  than  extracellular  concentration, 
particularly in fatty tissues. 

If the parent compound is the toxicologically active species, it may be possible 
to draw some conclusions about the distribution of the substance from its target 
tissues. If the substance is a dye, colouration of internal organs can give evidence 
of distribution.  

Clear signs of any toxicity. For example, CNS effects indicate that the substance 
(and/or its metabolites) has distributed to the CNS. However, not all behavioural 
changes  indicate  that  the  substance  has  reached  the  CNS.  The  behavioural 
change may be due to discomfort caused by some other effect of the substance. 

If the substance is a cationic trace element, absorption is likely to be very low 
(< 1 %). Stable isotopes or radioisotopes should be used and background levels 
determined to prevent analytical problems and inaccurate recoveries. 

3.2.4.  Accumulation potential 

It is important to consider the potential of a substance to accumulate or to be retained within 
the  body  because,  with  gradual  build-up  with  successive  exposures,  the  body  burden  can  be 
maintained for long periods of time. 

Lipophilic substances have the potential to accumulate within the body if the dosing interval is 
shorter  than  four  times  the  whole-body  elimination  half-life.  Although  there  is  no  direct 
correlation between the lipophilicity of a substance and its biological half-life, substances with 
high log Kow values tend to have longer half-lives unless high clearance counterbalances their 
large volume of distribution. On this basis, there is the potential for highly lipophilic substances 
(log Kow > 4) to accumulate in individuals who are frequently exposed to the substance. Once 
the exposure stops, the concentration within the body will decline at a rate determined by the 
half-life  of  the  substance.  Other  substances  that  can  accumulate  within  the  body  include 
substances that bind to endogenous proteins, and certain metals and ions that interact with the 
matrix of the bone (Slitt, 2018). 

Table 3 provides an overview of data that can be considered for estimating the accumulation of 
substances that migrate into the drinking water (oral exposure). 

 
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Table 3. How data from different sites can be used to interpret accumulation after oral 
exposure  

Site 

Characteristics of substance of concern 

Adipose tissue 

Lipophilic  substances  will  tend  to  concentrate  in  adipose  tissue  and  may 
accumulate. If the interval between exposures is less than four times the whole-
body half-life of the substance, then there is the potential for the substance to 
accumulate. Generally, substances with high log Kow values have long biological 
half-lives. On this basis, daily exposure to a substance with a log Kow of ≥ 4 could 
result in a build-up of the substance within the body. A substance with a log Kow 
of  ≤ 2  would  be  unlikely  to  accumulate  with  repeated  intermittent  exposure 
patterns  but  may  accumulate  if  exposures  are  continuous  or  there  is  limited 
excretion. Once exposure to the substance stops, the substance will be gradually 
eliminated at a rate dependent on the half-life of the substance. If fat reserves 
are mobilised more rapidly than normal, for example if an individual or an animal 
is  under  stress  or  lactating,  there  is  the  potential  for  higher  quantities  of  the 
parent compound to be released into the blood. 

Bone 

Certain metals, such as lead, and small ions, such as fluoride, can interact with 
ions in the matrix of bone. This interaction can displace the normal constituents 
of bone, leading to retention of the metal or ion.  

3.2.4.1.  Metabolism 

The main reason animal species toxicity and route-specific toxicity vary is the difference in the 
metabolism  of  substances  among  species  and  tissues.  The  liver  has  the  greatest  capacity  for 
metabolism  and  commonly  causes  route-specific  pre-systemic  effects  (first  pass),  particularly 
following  oral  intake.  However,  route-specific  (oral,  for  substances  that  migrate  into  drinking 
water) toxicity may  result from  several phenomena,  such as hydrolysis within the GI tract  or 
metabolism  by  GI  flora  or  within  the  GI  tract  epithelia  (mainly  in  the  small  intestine)  (for  a 
review, see Rourke and Sinal, 2014). 

Predicting  the  changes  that  a  substance  may  undergo  is  difficult  based  on  physico-chemical 
information  alone.  Although  it  is  possible  to  look  at  the  structure  of  a  molecule  and  identify 
potential metabolites, it is not certain that these reactions will occur in vivo (e.g. the molecule 
may not reach the necessary site for a particular reaction to take place). It is even more difficult 
to predict the extent to which it will be metabolised along different pathways and what animal 
species differences may exist. Consequently, experimental data can help in the assessment of 
potential metabolic pathways. Data on TK parameters can be derived from in vivo studies and, 
to  a  limited  extent,  from  standardised  in  vitro /  ex  vivo  studies  (e.g.  the  hepatic  clearance 
assay). 

3.2.4.2.  Excretion 

The  major  routes  of  substance  excretion  from  the  systemic  circulation  are  via  urine  and/or 
faeces, via bile and directly from the GI mucosa (see Slitt, 2018). 

The excretion processes in the kidney are passive glomerular filtration through membrane pores 
and active tubular secretion via carrier processes. Substances that are excreted in the urine tend 
to be water soluble and of low molecular weight (< 300 g/mol or Da in rats, mostly anionic and 
cationic  compounds)  and  are  generally  conjugated  metabolites  (e.g.  glucuronides,  sulphates, 
glycine conjugates) from phase II biotransformation. The kidneys will filter most of them out of 
the blood, although a small amount may enter the urine directly by passive diffusion, and there 
is  the  potential  for  reabsorption  into  the  systemic  circulation  across  the  tubular  epithelium. 

 
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Biliary  excretion  involves  active  secretion  rather  than  passive  diffusion.  Substances  that  are 
excreted into the bile tend to have a higher molecular weight. It has been found that, in rats, 
substances with a molecular weight of < ca 300 g/mol (or Da) do not tend to be excreted into 
the bile (Renwick, 1994). Threshold values for molecular weights of organic anions have been 
set  to  400 Da  in  rats  and  475 Da  in  humans,  whereas  no  threshold  values  can  be  set  for 
molecular weights of cations or cation/neutral compounds (Yang et al., 2009). Hepatic function 
highly influences the excretion of compounds via bile, as the metabolites formed in the liver may 
be excreted directly into the bile without entering the bloodstream. Blood flow is therefore an 
additional determining factor. 

Substances in the bile pass through the intestines before they are excreted in the faeces. As a 
result, the substances may undergo enterohepatic recycling (i.e. circulation of bile from the liver, 
where  it  is  produced,  to  the  small  intestine,  where  it  aids  the  digestion  of  fats  and  other 
substances, and back to the liver), which will prolong their biological half-life. This is a particular 
problem, although uncommon, for conjugated molecules that are hydrolysed by GI bacteria to 
form smaller, more lipid-soluble molecules that can be reabsorbed from the GI tract. Substances 
with strong polarity and high molecular weight are less likely to recirculate. Other substances 
excreted in the faeces are those that have diffused out of the systemic circulation into the GI 
tract directly, substances that have been removed from the GI mucosa by efflux mechanisms, 
and non-absorbed substances that have been ingested or inhaled and subsequently swallowed. 
However, depending on the possible metabolic changes, the compound that is finally excreted 
may have few or none of the physico-chemical characteristics of the parent compound. 

Table 4  provides  an  overview  of  the  data  that  can  be  used  for  estimating  the  excretion  of 
substances that migrate into drinking water (oral exposure). 

Table 4. How physico-chemical data can affect excretion after oral exposure 

Route 

Physico-chemical characteristics favourable for excretion 

Urine 

Bile 

Breast milk 

Saliva/sweat 

Hair/nails 

Exfoliation 

Characteristics favourable for urinary excretion in rats are a low molecular weight of 
< 300 g/mol (or Da), high water solubility and ionisation of the molecule at the pH of 
urine. 

In rats, molecules that are excreted in the bile are amphipathic (containing both polar 
and non-polar regions) and hydrophobic/non-polar and have a high molecular weight. 
In  general,  it  is  unlikely  that  in  rats  more  than  5–10 %  of  organic  cations  with  a 
molecular  weight  of  < 300 g/mol  (or  Da)  will  be  excreted  in  the  bile,  however,  for 
some  organic  cations  (e.g.  quaternary  ammonium  ions),  this  cut-off  may  be  even 
lower. Substances excreted in bile may undergo enterohepatic circulation. Substances 
with strong polarity and high molecular weight are less likely to recirculate. Little is 
known about the determinants of biliary excretion in humans. 

Substances present in plasma generally may also be found in breast milk. Lipid-soluble 
substances  may  be  present  at  higher  concentrations  in  milk  than  in  blood/plasma. 
Although lactation is a minor route of excretion, exposure of neonates via nursing may 
have toxicological significance for some substances. 

Non-ionised and small lipid-soluble molecules may be excreted in saliva or sweat. In 
saliva, the molecules may be swallowed. 

Metal ions may be incorporated into hair and nails. 

Highly lipophilic substances that penetrated the stratum corneum but did not penetrate 
the viable epidermis may be sloughed off with skin cells. 

 
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3.2.5.  Generating and integrating toxicokinetic information 

In  vivo  studies  provide  an  integrated  perspective  on  the  relative  importance  of  different 
processes in an intact biological system, which can be used for comparison with the results of 
toxicity studies. To ensure a valid set of TK data, an in vivo TK study has to consist of several 
experiments that include blood/plasma kinetics, mass balances and excretion experiments, as 
well  as  tissue  distribution  experiments.  Depending  on  the  problem  to  be  solved,  certain 
experiments  (e.g.  plasma  kinetics)  may  be  sufficient  to  provide  the  data  needed  for  further 
assessments (e.g. bioavailability). 

The high dose level administered in an ADME study should be linked to the dose levels that cause 
adverse effects in toxicity studies. Ideally, there should also be a dose without toxic effect, which 
should  be  in  the  range  of  expected  human  exposure.  A  comparison  of  kinetics  between  toxic 
dose  levels  and  dose  levels  that  are  likely  to  represent  human  exposure  values  may  provide 
valuable  information  for  the  interpretation  of  adverse  effects  and  is  thus  essential  for 
extrapolation. 

In an in vivo study, the systemic bioavailability is usually estimated by comparing either dose-
corrected  amounts  excreted  or  dose-corrected  AUCs  of  plasma/blood/serum  kinetic  profiles, 
after extravascular (e.g. oral) and intravascular (e.g. intravenous) administration. The systemic 
bioavailability is the dose-corrected amount excreted or AUC determined after an extravascular 
substance  administration,  divided  by  the  dose-corrected  amount  excreted  or  AUC  determined 
after an intravascular substance application, which corresponds, by definition, to a bioavailability 
of 100 %. This is valid only if the kinetics of the compound is linear (i.e. dose proportional) and 
relies on the assumption that the clearance is constant between experiments. If the kinetics is 
not linear, the experimental strategy has to be revised on a case-by-case basis, depending on 
the type of non-linearity involved (e.g. saturated protein binding, saturated metabolism). 

3.2.6.  Other methods for generating absorption, distribution, metabolism 

and excretion data 

In  addition  to  the  predictive  approaches  described  earlier  and  the  test  methods  described  in 
Section 6.5  of  DWD  Guidance  Volume I, 
in  vitro  studies  and  physiologically  based 
TK/pharmacokinetic modelling should also be considered for generating ADME data. 

3.2.6.1.  In vitro studies 

Generally, in vitro studies provide data on specific aspects of toxicokinetics, such as metabolism 
or  dermal  absorption.  A  major  advantage  of  in  vitro  studies  is  that  it  is  possible  to  carry  out 
parallel tests on different species, including humans, thus facilitating interspecies comparisons 
(e.g.  metabolite  profile,  metabolic  rate  constants).  In  recent  years,  methods  using  the 
appropriate physiologically based TK/pharmacokinetic models to integrate a number of in vitro 
results  into  a  prediction  of  ADME in  vivo  have  been  developed.  Such  methods  allow  both  the 
prediction of in vivo kinetics in humans and the progressive integration of all available data into 
a predictive model of ADME. The uncertainty associated with the prediction depends largely on 
the amount of available data. 

In  particular,  generating  TK  data  should  aim  to  provide  essential  information  for  building 
physiologically based kinetic models, to enable more accurate estimation of internal exposure, 
where relevant. 

 
 
 
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43 

3.2.6.2.  In silico methods: kinetic modelling 

An overview of in silico methods and guidance for use in TK assessment is provided in Guidance 
on  the  Biocidal  Products  Regulation –  Volume III  human  health –  Assessment  &  evaluation 
(Parts B+C) ( 36). Additional guidance has been developed by the OECD on the characterisation, 
validation and reporting of physiologically based kinetic models for regulatory purposes (Series 
on Testing and Assessment, No 331). This guidance document builds on previous documents on 
best practice in physiologically based kinetic model development and application, including the 
US  EPA  document  Approaches  for  the  application  of  physiologically  based  pharmacokinetic 
(PBPK) models and supporting data in risk assessment (2006), the International Programme on 
Chemical Safety (IPCS) guidance document Characterization and application of physiologically 
based pharmacokinetic models in risk assessment (2010) and EFSA’s Scientific opinion on good 
modelling  practice  in  the  context  of  mechanistic  effect  models  for  risk  assessment  of  plant 
protection products (2014). However, such modelling is still very uncommon. 

3.2.7.  Variability and uncertainty in toxicokinetics 

Uncertainty and variability are inherent to TK studies and potentially affect the conclusions of 
these studies. It is necessary to minimise uncertainty in order to assess the variability that may 
exist  between  individuals  so  that  there  is  confidence  in  the  TK  results  such  that  they  can  be 
useful for risk analysts and decision-makers. 

Variability typically refers to differences in the physiological characteristics among individuals 
(interindividual variability) or across time within a given individual (intraindividual variability). It 
may stem from genetic differences, activity level, lifestyle, physiological status, age, sex, etc. 
Variability is characteristic of animal and human populations. It can be observed and registered 
as information about the population, but it cannot be reduced. An important feature of variability 
is that it does not tend to decrease when larger samples of a population are examined. 

Variability  in  the  population  should,  then,  be  considered  in  TK  studies.  The  application  of 
probability  distributions  to  the  parameters  representing  the  distribution  of  physiological 
characteristics  in  the  population  may  introduce  variability  into  physiologically  based  kinetic 
models. The propagation of the variability to model predictions may be evaluated using Monte 
Carlo simulation methods. 

Uncertainty  can  be  defined  as  the  inability  to  make  precise  and  unbiased  statements.  It  is 
essentially the result of a lack of knowledge. Uncertainty in terms of information may decrease 
with the size of the sample studied. Further optimised experiments and better understanding of 
the process under study can theoretically eliminate or at least reduce uncertainty. 

Uncertainty may be related to the experimental nature of the data, the modelling procedure or 
the high inherent variability of biological systems. 

3.2.8.  Metabolism studies as a basis for internal dose considerations 

Biotransformation  of  a  substance  produces  metabolites  that  may  have  different  toxicological 
properties  than  the  substrate  from  which  they  are  formed.  Although  metabolism  is  generally 
considered to have a detoxification purpose, there are also many examples where metabolites 
have  higher  intrinsic  toxicity  than  the  parent  compound  itself  (bioactivation).  Therefore,  it  is 
necessary to know if the test substance is metabolised and to which metabolites. This enables 

(36)

https://echa.europa.eu/documents/10162/2324906/biocides_guidance_human_health_ra_iii_part_bc_en.pdf/

30d53d7d-9723-7db4-357a-ca68739f5094. 

 
 
 
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the assessment of results from toxicity studies in respect of grouping approaches and defines 
the internal dose (see Figure 3). 

Figure 3. Use of increasing knowledge on substance metabolism 

Is the test substance metabolised ?
Is the test substance metabolised ?

no
no

yes
yes

Parent compound
Parent compound
is relevant internal
is relevant internal
dose metric
dose metric

Metabolites known?
Metabolites known?

no
no

yes
yes

Identification via
Identification via
in vitro / in vivo meta-
in vitro / in vivo meta-
bolism studies
bolism studies

Tox. information on 
Tox. information on 
metabolites
metabolites
available ? (with respect
available ? (with respect
to species specificities)
to species specificities)

no
no

yes
yes

Perform studies 
Perform studies 
of metabolites
of metabolites
(include grouping
(include grouping
approach)
approach)

Define relevant 
Define relevant 
internal dose metric
internal dose metric

Apply in RA
Apply in RA

If  the  test  substance  is  not  metabolised,  the  parent  compound  is  the  relevant  marker  for 
measuring and defining the internal dose. If the test substance is metabolised, the knowledge 
of which metabolites are formed is essential for any further steps in an assessment. When this 
information  is  not  available,  it  can  be  investigated  by  appropriate  in  vitro  and/or  in  vivo 
metabolism studies. In special cases, metabolites may show a high degree of isomeric specificity, 
and this should be kept in mind when designing and interpreting mixtures of isomers, including 
racemates.  If  the  metabolites  are  known  and  the  toxicity  studies  are  available  for  these 
metabolites, the risk assessment may be carried out based on these data, and an assessment 
based on the definition of the internal dose can be made. If the toxicity profile of the metabolites 
is  unknown,  studies  that  address  the  metabolites’  toxicity  may  be  performed,  with  special 
consideration  given  to  the  potential  of  group  approaches,  particularly  if  a  substance  is  the 
metabolite  of  different  compounds,  for  example  carboxylic  acid  as  a  metabolite  of  different 
esters. 

 
 
 
 
 
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45 

TK information can be very helpful in bridging various gaps encountered across the whole risk 
assessment,  from  toxicity  study  design  and  biomonitoring  set-up (37)  to  the  derivation  of  the 
threshold  levels  and  various  extrapolations,  as  are  usually  needed  (cross-dose,  cross-species 
(including  human),  cross-exposure  regimen,  cross-route  and  cross-substance  extrapolations). 
The  internal  dose  is  the  central  output  parameter  of  TK  studies  and  therefore  the  external 
exposure–internal dose concept is broadly applicable in the various extrapolations mentioned. 
If, for that purpose, route-to-route extrapolation is necessary, and if assessment of combined 
exposure (via different routes) is needed, for systemic effects, internal exposure may have to 
be estimated. 

Exposure should normally be understood as an external exposure, which, in the context of the 
DWD, can be defined as the amount of substance ingested. 

Determination of the level of systemic exposure is considered synonymous with determination 
of  the  substance  bioavailability  to  the  general  circulation.  It  should  be  emphasised  that  it  is 
usually not possible to show that after oral ingestion the amount of a substance bioavailable is 
zero; however, in some cases, it may be predicted to be very low. The degree of certainty of the 
prediction will depend on the case. Important factors include the accuracy and reliability of the 
in  vivo,  in  vitro  or  in  silico  model  used,  the  performance  of  the  methods  used  to  assay  the 
substance or its metabolites, and the estimated variability in the target population. It should be 
noted that when a very low bioavailability is predicted, the degree of certainty of this prediction 
should be very high. 

The  compound’s  tissue  distribution  characteristics  can  be  an  important  determinant  of  its 
potential to cause toxicity in specific tissues. In addition, tissue distribution may be an important 
determinant of the ability of a compound to accumulate upon repeated exposure. However, the 
accumulation is substantially modified by the rate at which the compound is cleared. Correlation 
of  tissue  distribution  with  target  tissues  in  toxicity  studies  should  be  accomplished  while 
substantial amounts of the substance remain present in the body (e.g. around one or more times 
the peak blood concentration following oral absorption). Such data should quantify the parent 
compound and the metabolites to the extent feasible. If the metabolites are unknown or difficult 
to quantify, subtracting the parent compound from total radioactivity will provide an estimate of 
the behaviour of the total metabolites formed. 

3.2.9. 

Extrapolation 

For ethical reasons, if data allowing model parameters to be estimated are of poor quality and 
sparse, and do not often concern human populations,  recourse to  extrapolation is needed. In 
most cases TK data are gathered for a limited number of different concentrations (usually < 5 
different  concentrations)  and  exposure  times.  However,  risk  evaluation  should  assess  the 
different doses (concentrations and exposure times). Interdose/interexposure time extrapolation 
is a common way to satisfy this demand (Clewell and Andersen, 1996). 

In the rare cases where data on human volunteers are available, they concern only a very limited 
number  of  subjects.  Extrapolation  to  other  populations  can  be  done  (interindividual 
extrapolation). The problem of sensitive populations also arises, and hence a TK study should 
assess  different  genders,  ages,  ethnic  groups,  etc.  As  it  is  nearly  impossible  to  control  the 
internal dose in humans in practice, an alternative animal study is  often proposed. Since risk 

(37)  Biological  monitoring  information  should  be  seen  as  equivalent  to  (i.e.  as  having  neither  greater  nor  lesser 
importance than) other forms of exposure data. It should also be remembered that biological monitoring results 
reflect  an  individual’s  total  exposure  to  a  substance  from  any  relevant  route,  that  is,  from  consumer  products 
and/or  the  environment,  and  not  just occupational  exposure. Data  from  controlled  human  exposure  studies  are 
even less likely to be available. This is due to the practical and ethical considerations involved in deliberate exposure 
of individuals. 

 
 
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assessment  aims  to  protect  human  populations,  interspecies  extrapolation  (Davidson  et  al., 
1986; Watanabe and Bois, 1996) should be done. For practical reasons, the administration route 
in an experimental study can be different from the most likely exposure route. In this case, risk 
assessment suggests deciding on a route other than the one experimentally studied. Interroute 
extrapolation should be performed. 

Default  values  have  been  derived  to  match  the  extrapolation  idea  in  a  general  way.  The 
incorporation  of  quantitative  data  on  interspecies  differences  or  human  variability  in 
toxicokinetics and toxicodynamics into the dose–response / concentration–response assessment 
may be implemented through the development of substance-specific assessment factors (AF). 
The  use  of  substance-specific  AFs  may  improve  risk  assessment  of  individual  substances. 
Currently, data relevant for consideration are often restricted to the component of uncertainty 
related  to  interspecies  differences  in  toxicokinetics.  At  the  present  time,  there  are  commonly 
fewer data to address interspecies differences in toxicodynamics and interindividual variability 
in toxicokinetics and toxicodynamics. It is anticipated that the availability of such information 
will increase with a better common understanding of its appropriate nature (IPCS, 2001). The 
types of TK information that could be used include the rate and extent of absorption, the extent 
of  systemic  availability,  the  rate  and  extent  of  pre-systemic  (first-pass)  and  systemic 
metabolism, the extent of enterohepatic recirculation, information on the reactive metabolites 
formation  and  possible  species  differences,  and  knowledge  of  the  half-life  and  potential  for 
accumulation under repeated exposure. 

The need for these extrapolations can lead to more frequent use of physiological TK models than 
empirical  models  (Davidson  et  al.,  1986;  Watanabe  and  Bois,  1996).  Physiologically  based 
kinetic  models  facilitate  the  required  extrapolations  (interspecies,  intersubjects,  etc.).  For 
example,  by  changing  anatomical  parameters  such  as  organ  volumes  or  blood  flows,  a 
physiologically based kinetic model can be transposed from rat to human. 

3.2.9.1.  Interspecies extrapolation 

The use of animal data for toxicological risk assessment raises the question of how to extrapolate 
experimentally observed kinetics to human subjects or populations. The ability to compare data 
from animals and humans enables the definition of substance-specific interspecies extrapolation 
factors to replace the default values. One option is extrapolation based on different body sizes, 
which calculates the allometric factors. 

Allometric scaling is a commonly employed extrapolation approach. It is based on the principle 
that  biological  diversity  is  largely  explained  by  body  size  (Schneider  et  al.,  2004).  Allometric 
scaling  captures  the  correlations  of  physiological  parameters  or  toxicokinetics  with  body  size. 
More precisely, allometric equations relate the quantity of interest (e.g. a tissue dose) to a power 
function of body mass fitted across species, as follows: 

Y = a BWb 

where Y is the quantity of interest, a is the species-independent scaling coefficient ( 38),BW is 
body weight and b is the allometric exponent. 

The value of b depends on whether the quantity of interest scales approximately with body mass 
(b = 1), metabolic rate (39) (b = 0.75) or body surface area (b = 0.67 ( 40)) (Fiserova-Bergerova 

(38)  Fits single data points together to form an appropriate curve. 

(39)  In this context, it is not metabolism of compounds. The factor adapts different levels of oxygen consumption. 

(40)  This scaling factor is generally justified on the basis of the studies by Freireich et al. (1966), who examined the 

interspecies differences in the toxicity of a variety of antineoplastic drugs. 

 
 
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and Hugues, 1983; Davidson et al., 1986; West et al., 1997). As it is easy to apply the allometric 
scaling, it is probably the most convenient approach to interspecies extrapolation. However, it 
is  very  approximate  and  may  not  hold  for  the  substance  of  interest.  It  can  therefore  be 
considered a default approach to be used only in the absence of specific data on the relevant 
chemical species of interest. 

For  a  substance  that  demonstrates  significant  interspecies  variation  in  animal  toxicity 
experiments, the most susceptible animal species are generally used as the reference point for 
extrapolation. The need for additional uncertainty factors should be evaluated case by case. 

To better estimate tissue exposure across species, physiologically based kinetic models may be 
used for the toxicant of interest (Watanabe and Bois, 1996). These models account for transport 
mechanisms and metabolism within the body. The same equation set then models the processes 
for  all  species  considered.  Differences  between  species  are  assumed  to  be  due  to  different 
(physiological, chemical and metabolic) parameter values. Extrapolation of physiologically based 
kinetic  models  then  relies  on  replacing  the  model  parameter  values  of  one  species  with  the 
parameter  values  of  the  species  of  interest.  For  physiological  parameters,  numerous  studies 
(Arms and Travis, 1988; Brown et al., 1997; ICRP, 2002) give standard parameter values for 
many species. Chemical (log P) and metabolic parameter values are usually less easily found. 
When  the  parameter  values  of  the  physiologically  based  kinetic  model  are  not  known  for  the 
species of interest, the option of in vitro data, (Q)SAR predictions or allometric scaling of those 
parameters  is  still  possible.  To  consider  population  variability  in  the  extrapolation  process, 
probability  distributions  of  parameters  may  be  used  rather  than  single  parameter  values. 
Physiologically based kinetic models can be particularly useful where data are being extrapolated 
to  population  subgroups  for  which  only  little  information  is  available,  for  example  pregnant 
women or infants (Luecke et al., 1994; Young et al., 2001). 

3.2.9.2.  Interroute extrapolation 

As explained in Chapter 5, the oral route is the most relevant one for substances migrated in 
drinking water. If studies with a route of exposure other than the oral route are used, route-to-
route extrapolation should be applied. 

Route-to-route  extrapolation  is  defined  as  the  prediction  of  the  total  amount  of  a  substance 
administered by one route that would produce the same systemic toxic response as that obtained 
for a given amount of a substance administered by another route. 

In general, route-to-route extrapolation is considered to be a poor substitute for toxicity data 
obtained using the appropriate route of exposure. Uncertainties in extrapolation increase when 
performing risk assessment with toxicity data obtained by an administration route that does not 
correspond to the human route of exposure. 

Because of the high level of uncertainty in extrapolation, it is recommended that inhalation and, 
in  particular,  dermal  studies  not  be  used  for  hazard  assessment  and  risk  acceptance  for  the 
purpose of the DWD. However, if a study performed via a different route of administration (e.g. 
inhalation) is used, route-to-route extrapolation has to be considered, taking into account all the 
uncertainties mentioned below. 

When route-to-route extrapolation is used, the following aspects should be carefully considered. 

•  Nature of the effect. Route-to-route extrapolation is applicable only for the evaluation of 
systemic effects. For the evaluation of local effects after repeated exposure, only results from 
toxicity studies performed with the route under consideration can be used. 

•  TK data (ADME). The major factors responsible for differences in toxicity due to route of 

exposure include: 

 
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differences in bioavailability or absorption; 

differences in metabolism (first-pass effects); 

differences in internal exposure pattern (i.e. internal dose). 

48 

o 

o 

o 

In  the  absence  of  relevant  kinetic  data,  route-to-route  extrapolation  is  possible  only  if  the 
following assumptions are reasonably valid: 

•  absorption can be quantified; 

• 

• 

toxicity is a systemic effect, not a local one (the compound is relatively soluble in body fluids, 
therefore systemically bioavailable), and internal dose can be estimated; 

first-pass effects are minimal. 

For  further  guidance  on  how  to  perform  route-to-route  extrapolation,  please  follow  the 
recommendations  provided  in  ECHA’s  Guidance  on  Information  Requirements  and  Chemical 
Safety Assessment – Chapter R.8: Characterisation of dose [concentration]–response for human 
health (41). 

3.3.  Repeated dose toxicity 

The sections on repeated dose toxicity, neurotoxicity, endocrine disruption and immunotoxicity 
in  DWD  Guidance  Volume I  (Sections 6.6  and  6.10)  should  be  considered  together  with  the 
elements  described  in  this  section  for  the  assessment  of  repeated  dose  toxicity.  Information 
from experimental and non-test approaches with regard to other end points (e.g. toxicokinetics 
and genotoxicity) should be assessed using a WoE approach in the assessment of toxicological 
findings  following  repeated  dose  administration.  The  ultimate  goal  is  to  identify  the  potential 
mode of action and underlying key events. 

3.3.1.  Definition of repeated dose toxicity 

Repeated dose toxicity comprises the adverse general (i.e. excluding reproductive, genotoxic or 
carcinogenic effects) toxicological effects occurring as a result of repeated daily dosing with or 
exposure to a substance for a part of the expected lifespan (subacute or subchronic exposure) 
or for a major part of the lifespan, in the case of chronic exposure. 

The term general toxicological effects (hereafter referred to as general toxicity) includes effects 
on  body  weight  and/or  body  weight  gain,  absolute  and/or  relative  organ  and  tissue  weights, 
alterations  in  clinical  chemistry,  urinalysis  and/or  haematological  parameters,  functional 
disturbances in the nervous system as well as in organs and tissues in general, and pathological 
alterations  in  organs  and  tissues  as  examined  macroscopically  and  microscopically.  Repeated 
dose toxicity studies may also examine parameters that have the potential to identify specific 
manifestations  of  toxicity,  such  as  neurotoxicity,  immunotoxicity,  endocrine-mediated  effects, 
reproductive toxicity and carcinogenicity. 

An  adverse  effect  is  a  change  in  the  morphology,  physiology,  growth,  development, 
reproduction or lifespan of an organism, system or (sub)population that results in an impairment 
of functional capacity, an impairment of the capacity to compensate for additional stress or an 
increase in susceptibility to other influences. 

(41)  https://echa.europa.eu/documents/10162/17224/information_requirements_r8_en.pdf/e153243a-03f0-44c5-

8808-88af66223258?t=1353935239897. 

 
 
 
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A chemical substance may induce systemic and/or local effects, defined as follows: 

•  a local effect is an effect that is observed at the site of first contact, caused irrespective of 

whether a substance is systemically available; 

•  a systemic effect is an effect that is normally observed distant from the site of first contact, 
that is, after having passed through a physiological barrier (mucous membrane of the GI 
tract or the respiratory tract, or the skin) and becomes systemically available. 

It should be noted, however, that toxic effects on surface epithelia may reflect indirect effects 
as  a  consequence  of  systemic  toxicity  or  secondary  effects  as  a  consequence  to  systemic 
distribution of the substance or its active metabolite(s). 

Repeated dose toxicity tests provide information on possible adverse effects likely to arise from 
repeated exposure of target organs and information on dose–response relationships. 

The  determination  of  the  dose–response  relationship  should  lead  to  the  identification  of  the 
NOAEL/LOAEL/BMD. As part of the risk assessment process for relevant chemical species, data 
on the adverse effects a substance may cause and the dose levels at which the effects occur are 
evaluated in the light of the likely extent of human exposure to the relevant chemical species 
via  water  intended  for  human  consumption,  so  that  the  potential  risk(s)  to  health  may  be 
ascertained. 

The objectives of assessing repeated dose toxicity are to evaluate: 

•  whether exposure of humans to a substance has been associated with adverse toxicological 
effects occurring as a result of repeated daily exposure for a part of the expected lifespan 
or for a major part of the lifespan; 

•  whether administration of a substance to experimental animals causes adverse 

toxicological effects as a result of repeated daily exposure for a part of the expected 
lifespan or for a major part of the lifespan – effects that may be predictive of possible 
adverse human health effects; 

• 

• 

• 

the target organs, potential cumulative effects and the reversibility of the adverse 
toxicological effects; 

the dose–response relationship and threshold for any of the adverse toxicological effects 
observed in the repeated dose toxicity studies; 

the basis for risk characterisation and classification and labelling of substances for systemic 
target organ toxicity. 

3.3.2.  Data to be used in the hazard assessment 

3.3.2.1.  Non-human data for repeated dose toxicity 

3.3.2.1.1. 

Non-testing data for repeated dose toxicity 

See Section 3.1.2.2.5 (on (Q)SARs) and Section 3.1.3 (on the read-cross approach). 

3.3.2.1.2. 

Testing data for repeated dose toxicity 

In vitro data. While several assays measuring repeated in vitro exposure of, for example, liver, 
kidney and brain cells are under development, currently the in vitro data cannot be used on their 

 
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own in regulatory decisions such as risk assessment. However, such data may be helpful in the 
assessment of repeated dose toxicity, for instance to detect local target organ effects and/or to 
clarify the mechanisms of action. Since, at present, there are no validated in vitro methods that 
have received regulatory acceptance, the quality of each of these studies and the adequacy of 
the data provided should be carefully evaluated. 

Generic guidance is given in ECHA’s Guidance on Information Requirements and Chemical Safety 
Assessment – Chapter R.4: Evaluation of available information ( 42) and Guidance on Information 
Requirements  and  Chemical  Safety  Assessment –  Chapter R.5:  Adaptation  of  information 
requirements (43),  for  judging  the  applicability  and  validity  of  the  outcome  of  various  study 
methods and assessing the quality of the conduct of a study and the reproducibility of data and 
aspects such as vehicle, number of replicates, exposure/incubation time, GLP compliance and 
comparable quality description. 

Animal  data.  The  most  appropriate  data  on  repeated  dose  toxicity  for  use  in  hazard 
characterisation  and  risk  assessment  of  relevant  chemical  species  identified  in  the  migration 
water  are  primarily  obtained  from  oral  studies  in  experimental  animals  conforming  to 
internationally agreed test guidelines. In some circumstances, repeated dose toxicity studies not 
conforming to conventional test guidelines may also provide  relevant  information for this  end 
point. 

The  information  that  can  be  obtained  from  the  available  EU/OECD  test  guideline  studies  for 
repeated  dose  toxicity  is  briefly  summarised  below.  Table 5  summarises  the  parameters 
examined  in  these  OECD  test  guideline  studies  in  more  detail  to  facilitate  overview  of  the 
similarities and differences between the various studies. 

Repeated dose 28-day toxicity oral study 

The relevant guideline for this end point is EU B.7 / OECD TG 407. The 28-day studies provide 
information on the toxicological effects arising from exposure to the substance during a relatively 
limited period of the animal’s lifespan. 

Repeated dose 90-day oral toxicity studies 

Separate guidelines are available for studies using oral administration (OECD TG 408 / EU B.26 
and OECD TG 409 / EU B.27 in  rodent and non-rodent species,  respectively). The principle of 
these study protocols is identical, although the revised OECD TG 408 (2018) protocol includes 
additional parameters enabling the identification of a neurotoxic potential, immunological effects 
or reproductive organ toxicity. 

The  90-day  study  provides  information  on  the  general  toxicological  effects  on  target  organs 
arising from  subchronic exposure (a prolonged period of the animal’s  lifespan) covering post-
weaning maturation and growth well into adulthood. 

Chronic toxicity studies 

The chronic toxicity studies (OECD TG 452 / EU B.30) provide information on the toxicological 
effects arising from repeated exposure over a prolonged period of time covering a major part of 
the animal’s lifespan. The duration of the chronic toxicity studies should be at least 12 months. 

(42)  https://echa.europa.eu/documents/10162/17235/information_requirements_r4_en.pdf/d6395ad2-1596-4708-

ba86-0136686d205e?t=1323782558175. 

(43)  https://echa.europa.eu/documents/10162/17235/information_requirements_r5_en.pdf/51ffb7a7-baef-43ef-bac7-

501e95b5a1d5?t=1323783044916. 

 
 
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The  combined  chronic  toxicity /  carcinogenicity  studies  (OECD  TG 453 /  EU B.33)  include  an 
additional high-dose satellite group for evaluation of pathology other than neoplasia. The satellite 
group should be exposed for at least 12 months and the animals in the carcinogenicity part of 
the study should be retained in the study for the majority of the normal lifespan of the animals. 

Ideally,  the  chronic  studies  should  allow  for  the  detection  of  general  toxicity  effects 
(physiological, biochemical and haematological effects, etc.) but could also provide information 
on  neurotoxic,  immunotoxic,  reproductive  and  carcinogenic  effects  of  the  relevant  chemical 
species.  However,  in 12-month  studies,  it  is  possible  that  non-specific  life-shortening  effects, 
which require a long latent period or are cumulative, may not be detected in this study type. In 
addition, the combined study will allow for the detection of neoplastic effects and determination 
of carcinogenic potential and life-shortening effects. 

Repeated dose toxicity study combined with reproduction/developmental toxicity screening test 

The combined repeated dose toxicity / reproductive screening study (OECD TG 422 / EU B.64) 
provides information on the toxicological effects arising from repeated exposure (generally oral 
exposure) over a period of about 6 weeks for males and approximately 54 days for females (a 
relatively limited period of the animal’s lifespan) and information on reproductive toxicity. For 
the repeated dose toxicity part, OECD TG 422 / EU B.64 is in concordance with OECD TG 407 / 
EU B.7 except for use of pregnant females and the longer exposure duration in OECD TG 422 / 
EU B.64 than in OECD TG 407 / EU B.7. 

Neurotoxicity studies 

The neurotoxicity study in rodents (OECD TG 424 / EU B.43) is designed to further characterise 
potential  neurotoxicity  observed  in  repeated  dose  systemic  toxicity  studies.  The  neurotoxicity 
information  on  major  neurobehavioural  and 
study 
neuropathological effects in adult rodents. 

in  rodents  will  provide  detailed 

Delayed neurotoxicity studies of organophosphorus substances 

The delayed neurotoxicity study (OECD TG 419 / EU B.38) is specifically designed to be used in 
the assessment and evaluation of the neurotoxic effects of organophosphorus substances. This 
study provides information on the delayed neurotoxicity arising from repeated exposure over a 
relatively limited period of the animal’s lifespan. 

Other studies providing information on repeated dose toxicity 

Although  they  are  not  aimed  at  investigating  repeated  dose  toxicity  per  se,  other  available 
OECD/EU  test  guideline  studies  involving  repeated  exposure  of  experimental  animals  may 
provide useful information on repeated dose toxicity. These studies are summarised in Table 6. 

It should be noted that the repeated dose toxicity studies, if carefully evaluated, may provide 
information  on  potential  reproductive  toxicity  and  on  carcinogenicity  (e.g.  pre-neoplastic 
lesions). 

The one-generation, two-generation and extended one-generation reproductive toxicity studies 
(OECD TG 415 / EU B.34; TG 416 / EU B.35; TG 443 / EU B.56) may provide information on the 
general  toxicological  effects  arising  from  repeated  exposure  over  a  prolonged  period  of  time 
(about 90 days for parental animals), as clinical signs of toxicity, body weight, selected organ 
weights, and gross and microscopic changes of selected organs are recorded. 

The prenatal developmental toxicity study (OECD TG 414 / EU B.31), reproduction/developmental 
toxicity  screening  study  (OECD  TG 421 /  EU B.63)  and  developmental  neurotoxicity  study 
(OECD TG 426) may give some indications of general toxicological effects arising from repeated 

 
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exposure over a relatively limited period of the animal’s lifespan, as clinical signs of toxicity and 
body weight are recorded. 

The carcinogenicity study (OECD TG 451 / EU B.32) will, in addition to providing information on 
neoplastic lesions, provide information on the general toxicological effects arising from repeated 
exposure over a major portion of the animal’s lifespan, as clinical signs of toxicity, body weight, 
and gross and microscopic changes of organs and tissues are recorded. 

The  basic  concept  of  repeated  dose  toxicity  studies  to  generate  data  on  target  organ  toxicity 
following subacute to chronic exposure is to treat experimental animals for 4 weeks, 13 weeks 
or  longer.  In  addition,  other  studies  performed  in  experimental  animals  may  provide  useful 
information on repeated dose toxicity. While the majority of non-animal methods are currently 
in the research and development phase and not yet poised as substitutes for subchronic/chronic 
animal  studies,  there  is  the  potential  to  enhance  data  collection  for  risk  assessment.  This 
presents  opportunities  for  increased  efficiency,  reduced  animal  usage  and  more  effective 
resource utilisation. 

Consideration of in vitro data as well as TK data can be useful in the evaluation of the repeated 
dose toxicity information, as these data can assist in the correct derivation of internal exposure 
values,  the  assessment  of  human  relevance  and  the  correct  application  of  AFs  in  deriving 
threshold levels and in the design and development of new and more biologically relevant tests. 

The following general guidance is provided for the evaluation of repeated dose toxicity data and 
the development of the WoE; in this respect, all other information, including non-test methods, 
must be taken into account in the WoE building. 

•  Studies  on  the  most  sensitive  animal  species  should  be  selected  as  the  significant  ones, 
unless  TK  and  toxicodynamic  data  show  that  this  species  is  less  relevant  for  human  risk 
assessment. 

•  Studies  using  an  appropriate  route,  duration  and  frequency  of  exposure  in  relation  to  the 
expected  route(s),  frequency  and  duration  of  human  exposure  have  greater  weight.  For 
relevant chemical species identified in the migration water, those are oral subchronic and/or 
chronic repeated dose toxicity studies. 

•  Studies  enabling the identification  of the relevant point of departure  (NOAEL/LOAEL/BMD) 

and robust hazard identification have a greater weight. 

•  Studies of a longer duration should be given greater  weight than a repeated dose toxicity 
study of a shorter duration in the determination of the most relevant NOAEL (e.g. chronic 
study is preferred over subchronic). 

If  sufficient  evidence  is  available  to  identify  the  critical  effect(s)  (with  regard  to  the  dose–
response relationship(s) and the relevance for humans) and the target organ(s) and/or tissue(s), 
greater weight should be given to specific studies investigating this effect in the identification of 
the NOAEL. The critical effect can be a local as well as a systemic effect. 

While  data  available  from  repeated  dose  toxicity  studies  not  performed  in  accordance  with 
conventional guidelines and/or GLP may still provide information of relevance for risk assessment 
and classification and labelling, such data require extra careful evaluation. 

Data  from  non-guideline  studies  must  be  considered  to  be  equivalent  to  data  generated  by 
corresponding test methods if the following conditions are met: 

 
 
 
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• 

• 

they are adequate for the purpose of risk assessment; 

there  is  adequate  coverage  of  the  key  parameters  (e.g.  observations,  body  weight, 
food/water  consumption,  haematology  and  clinical  biochemistry,  gross  pathology  and 
histopathology) that the corresponding test guideline methods anticipate will be investigated; 

•  exposure duration is comparable to or longer than the corresponding test guideline method 

if exposure duration is a relevant parameter; 

•  adequate and reliable documentation of the study is provided. 

In all other situations, non-guideline studies may contribute to the overall weight of the evidence 
but  cannot  stand  alone  as  a  hazard  and  risk  assessment  of  a  relevant  chemical  species  and, 
thus, cannot serve as the sole basis for an assessment of repeated dose toxicity or for exemption 
from the standard information requirements for repeated dose toxicity. That is, they cannot be 
used to identify a relevant chemical species as being adequately controlled in relation to repeated 
dose toxicity. 

The  existing  information  is  considered  sufficient  when,  based  on  a  WoE  analysis,  the  critical 
effect(s) and target organ(s) and tissue(s) can be identified, the dose–response relationship(s) 
and NOAEL(s) and/or LOAEL(s) and/or BMD(s) for the critical effect(s) can be established, and 
the relevance for human beings can be assessed. 

It should be noted that potential effects in certain target organs (e.g. thyroid) following repeated 
exposure may not be observed within the span of shorter studies, for example the 28-day study. 
Attention  is  also  drawn  to  the  fact  that  the  protocols  for  the  28-day  and  90-day  oral  studies 
include  additional  parameters  compared  with  those  for  the  28-day  and  90-day  dermal  and 
inhalation protocols. 

Where  it  is  considered  that  the  existing  data  are  as  a  whole  inadequate  to  provide  a  clear 
assessment  of this  end point, the need for further testing  should be  considered in view  of all 
available  relevant  information  on  the  relevant  chemical  species,  including  use  pattern,  the 
potential for human exposure, physico-chemical properties and structural alerts. 

Specific investigations such as studies of neurotoxicity, endocrine disruption or immunotoxicity 
are also elements in the testing strategy that should be taken into account. 

Regarding neurotoxicity and immunotoxicity, standard 28-day and 90-day oral toxicity studies 
include end points capable of detecting such effects. Indicators of neurotoxicity include clinical 
functional  observational  battery,  motor  activity  assessment  and 
observations,  a 
histopathological examination of the spinal cord and sciatic nerve. Indicators of immunotoxicity 
include changes in haematological parameters and serum globulin levels, alterations in immune 
system organ weights such as the spleen and thymus, and histopathological changes in immune 
organs such as the spleen, thymus, lymph nodes and bone marrow. Where data from standard 
28-day and 90-day oral studies identify evidence of direct test substance-related adverse effects 
for neurotoxicity  or immunotoxicity, it is advised that those effects be  considered in the WoE 
approach  to  decide  on  the  need  for  further  testing.  It  should  be  noted  that  very  limited  end 
points indicative of neurotoxicity and immunotoxicity are examined in the standard 28-day and 
90-day dermal or inhalation repeated dose toxicity studies. 

Regarding endocrine disruptors (EDs), ECHA/EFSA guidance on the identification of EDs (44) is 
available  and  describes  how  to  gather,  evaluate  and  consider  all  relevant  information  for  the 

(44)  https://doi.org/10.2903/j.efsa.2018.5311. 

 
 
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assessment, conduct a mode of action analysis and apply a WoE approach, in order to establish 
whether the ED criteria are fulfilled. 

Furthermore, guidance to facilitate the interpretation of hazard data derived from screens and 
tests  in  the  OECD  conceptual  framework (45)  was  published  in  2012  and  updated  in  2018  to 
reflect new and updated OECD test guidelines.  

In general, results from toxicological studies requiring repeated administration of a substance, 
such  as  reproduction  and  developmental  toxicity  studies  and  carcinogenicity  studies,  can 
contribute  to  the  assessment  of  repeated  dose  toxicity.  However,  such  toxicological  studies 
rarely  provide  the  information  obtained  from  a  standard  repeated  dose  toxicity  study  and, 
therefore, cannot stand alone as the sole basis for the assessment of repeated dose toxicity or 
for exemption from the standard information requirements for repeated dose toxicity. 

Studies such as acute toxicity and in vivo genotoxicity studies contribute limited information to 
the  overall  assessment  of  repeated  dose  toxicity.  However,  such  studies  may  be  useful  in 
deciding on the dose levels for use in repeated dose toxicity studies. 

Guidance on the dose selection for repeated dose toxicity testing is provided in detail in the EU 
and OECD test guidelines. Unless limited by the physico-chemical nature or biological effects of 
the test substance, the highest dose level possible should be chosen, with the aim of inducing 
toxicity but not death or severe suffering. 

TK studies may be helpful in the evaluation and interpretation of repeated dose toxicity data, for 
example in relation to the accumulation of a substance or its metabolites in certain tissues or 
organs as well as the mechanistic aspects of repeated dose toxicity and species differences. 

Table 5. Overview of in vivo oral repeated dose toxicity test guideline studies  

Test 

Design 

End points 

OECD TG 407 (EU B.7): 
repeated dose 28-day oral 
toxicity study in rodents 

Exposure for 28 days 

Clinical observations 

At least three dose levels 
plus control 
At least five males and 
females per group 
Preferred rodent species: 
rat 

Functional observations (fourth exposure 
week – sensory reactivity to stimuli of 
different types, grip strength, motor 
activity) 

Body weight and food/water consumption 
Haematology (haematocrit, haemoglobin, 
erythrocyte count, total and differential 
leucocyte count, platelet count, blood 
clotting time/potential) 

Clinical biochemistry 

Urinalysis (optional) 

Gross necropsy (full, detailed, all animals) 
Organ weights (all animals – liver, kidneys, 
adrenals, testes, epididymides, thymus, 
spleen, brain, heart) 
Histopathology (full, at least control and 
high-dose groups – all gross lesions, brain, 
spinal cord, stomach, small and large 
intestines, liver, kidneys, adrenals, spleen, 
heart, thymus, thyroid, trachea and lungs, 
gonads, accessory sex organs, urinary 
bladder, lymph nodes, peripheral nerve, a 

(45)  http://www.oecd.org/env/ehs/testing/oecdworkrelatedtoendocrinedisrupters.htm#GD_Standardized_TG. 

 
 
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Test 

Design 

End points 

OECD TG 408 (EU B.26): 
repeated dose 90-day oral 
toxicity study in rodents 

section of bone marrow) 
Endocrine end points: T3, T4 and TSH 
(optional) 

Exposure for 90 days 

Clinical observations 

At least three dose levels 
plus control 

At least 10 males and 
females per group 

Preferred rodent species: 
rat 

Ophthalmological examination 

Functional observations (towards end of 
exposure period – sensory reactivity to 
stimuli of different types, grip strength, 
motor activity) 

Body weight and food/water consumption 

Haematology (haematocrit, haemoglobin, 
erythrocyte count, total and differential 
leucocyte count, platelet count, blood 
clotting time/potential) 

Clinical biochemistry 

Urinalysis 

Gross necropsy (full, detailed, all animals) 

Organ weights (all animals – liver, kidneys, 
adrenals, testes, epididymides, uterus, 
ovaries, thymus, spleen, brain, heart) 

Histopathology (full, at least control and 
high-dose groups – all gross lesions, brain, 
spinal cord, pituitary, thyroid, parathyroid, 
thymus, oesophagus, salivary glands, 
stomach, small and large intestines, liver, 
pancreas, kidneys, adrenals, spleen, heart, 
trachea and lungs, aorta, gonads, uterus, 
accessory sex organs, female mammary 
gland, prostate, urinary bladder, gall 
bladder (mouse), lymph nodes, peripheral 
nerve, a section of bone marrow, and 
skin/eyes on indication) 

Endocrine end points: T4 and thyroid 
weight (mandatory); T3, TSH and other 
hormone measurements (optional) 

Observations of the spermatogenesis and 
oestrous cycle 

OECD TG 409 (EU B.27): 
repeated dose 90-day oral 
toxicity study in non-
rodents 

Exposure for 90 days 

Clinical observations 

At least three dose levels 
plus control 

At least four males and 
females per group 

Preferred species: dog 

Ophthalmological examination 

Body weight and food/water consumption 

Haematology (as in OECD TG 408 / 
EU B.26) 

Clinical biochemistry 

Urinalysis 

Gross necropsy (full, detailed, all animals) 

Organ weights (as in OECD TG 408 / 
EU B.26 – additional: gall bladder, thyroid, 
parathyroid) 

Histopathology (as in OECD TG 408 / 
EU B.26 – additional: gall bladder, eyes) 

OECD TG 452 (EU B.30): 
chronic toxicity studies 

Exposure for at least 
12 months 

Clinical observations, including neurological 
changes 

 
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Test 

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Design 

End points 

OECD TG 453 (EU B.33): 
combined chronic toxicity / 
carcinogenicity studies 

OECD TG 422 (EU B.64): 
combined repeated dose 
toxicity study with the 
reproduction/developmental 
toxicity screening test 

At least three dose levels 
plus control 
Rodents: at least 20 
males and females per 
group 
Non-rodents: at least 
four males and females 
per group 
Preferred rodent species: 
rat 

Preferred non-rodent 
species: dog 

Exposure for at least 
12 months (satellite 
groups) or majority of 
normal lifespan 
(carcinogenicity part) 

At least three dose levels 
plus control 

At least 50 males and 
females per group 

Satellite group: at least 
20 males and females 
per group 

Preferred species: rat 

Exposure for a minimum 
of 4 weeks (males) or 
from 2 weeks prior to 
mating until at least 
postnatal day 4 
(females – at least 
6 weeks of exposure) 
At least three dose levels 
plus control 

Ophthalmological examination 

Body weight and food/water consumption 

Haematology (haematocrit, haemoglobin, 
erythrocyte count, total leucocyte count, 
platelet count, clotting potential) 

Clinical biochemistry 

Urinalysis 

Gross necropsy (full, detailed, all animals) 

Organ weights (all animals – brain, liver, 
kidneys, adrenals, gonads, 
thyroid/parathyroid (non-rodents only)) 

Histopathology (full, at least control and 
high-dose groups – all grossly visible 
tumours and other lesions, as well as 
essentially the same organs and tissues as 
in the 90-day studies (OECD TG 408/409 / 
EU B.29/B.27)) 

Essentially as in OECD TG 452 / EU B.30 

Clinical observations as in OECD TG 407 / 
EU B.7 
Functional observations as in OECD 
TG 407 / EU B.7 

Body weight and food/water consumption 

Haematology as in OECD TG 407 / EU B.7 

Clinical biochemistry 

Urinalysis (optional) 

At least 10 males and 
females per group 

Gross necropsy (full, detailed, all adult 
animals) 

Organ weights (testes and epididymides – 
all males; liver, kidneys, adrenals, thymus, 
spleen, brain, heart – in five animals of 
each sex per group, that is, as in OECD 
TG 407 / EU B.7) 

Histopathology (ovaries, testes, 
epididymides, accessory sex organs, all 
gross lesions – all animals in at least 
control and high-dose groups; brain, spinal 
cord, stomach, small and large intestines, 
liver, kidneys, adrenals, spleen, heart, 
thymus, thyroid, trachea and lungs, urinary 
bladder, lymph nodes, peripheral nerve, a 

 
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Test 

Design 

End points 

OECD TG 424 (EU B.43): 
neurotoxicity study in 
rodents 

Exposure for at least 
28 days 

Dose levels: not 
specified 

At least 10 males and 
females per group 
Preferred rodent species: 
rat 
Generally oral route of 
administration 

section of bone marrow – in five animals of 
each sex in at least control and high-dose 
groups, that is, as in OECD TG 407 / 
EU B.7) 

Endocrine end points: anogenital 
difference; nipple retention, T4 

Detailed clinical observations 

Functional observations (sensory reactivity 
to stimuli of different types, grip strength, 
motor activity, more specialised tests on 
indication) 

Ophthalmological examination 

Body weight and food/water consumption 

Haematology (haematocrit, haemoglobin, 
erythrocyte count, total and differential 
leucocyte count, platelet count, blood 
clotting time/potential) 

Clinical biochemistry 

Histopathology: at least five animals of 
each sex per group) for neuropathological 
examinations (brain, spinal cord and 
peripheral nerves); remaining animals to be 
used either for specific neurobehavioural, 
neuropathological, neurochemical or 
electrophysiological procedures that may 
supplement the histopathology or, 
alternatively, for routine pathological 
evaluations in accordance with the 
guidelines for standard repeated dose 
toxicity studies 

OECD TG 419 (EU B.38): 
delayed neurotoxicity of 
organophosphorus 
substances – 28-day 
repeated dose study 

Exposure for 28 days 

Detailed clinical observations 

At least three dose levels 
plus control 

At least 12 birds per 
group 

Body weight and food/water consumption 

Clinical biochemistry (neuropathy target 
esterase activity, acetylcholinesterase 
activity) 

Species: domestic laying 
hen 

Gross necropsy (all animals) 

Histopathology (neural tissue) 

NB: 
Repeated dose dermal and inhalation toxicity studies are not listed in the table. 

T3, thyroid hormone triiodothyronine; T4, thyroid hormone thyroxine; TSH, thyroid stimulating hormone. 

Table 6. Overview of other in vivo test guideline studies giving information on repeated dose 
toxicity  

Test 
OECD TG 416 (EU B.35): 
two-generation reproduction 
toxicity study 

Design 

End points (general toxicity) 

Exposure before 
mating for at least 
one spermatogenic 
cycle until weaning of 
second generation 
At least three dose 
levels plus control 
At least 20 parental 
males and females 
per group  

Clinical observations 
Body weight and food/water consumption 
Gross necropsy (all parental animals) 
Organ weights (reproductive organs, brain, 
liver, kidneys, spleen, pituitary, thyroid, adrenal 
glands and known target organs) 
Histopathology (reproductive organs, previously 
identified target organ(s) – at least control and 
high-dose groups) 

 
58 

Test 
OECD TG 415 (EU B.34): 
one-generation reproduction 
toxicity study 

OECD TG 443 (EU B.56): 
extended one-generation 
reproductive toxicity study 
OECD TG 414 (EU B.31): 
prenatal developmental 
toxicity study 

OECD TG 421 (EU B.63): 
reproduction/developmental 
toxicity screening test 

OECD TG 426 (EU B.53): 
developmental neurotoxicity 
study 

OECD TG 451 (EU B.32): 
carcinogenicity studies 

Design 

Exposure before 
mating for at least 
one spermatogenic 
cycle until weaning of 
first generation 
At least three dose 
levels plus control 
At least 20 parental 
males and females 
per group 
As described in OECD 
TG 443 / EU B.56 

Exposure at least 
from implantation to 
1 or 2 days before 
expected birth 
At least three dose 
levels plus control 
At least 20 pregnant 
females per group 
Exposure from 
2 weeks prior to 
mating until at least 
postnatal day 4 
At least three dose 
levels plus control 
At least 8–10 parental 
males and females 
per group 
Exposure at least 
from implantation and 
throughout lactation 
(postnatal day 20) 
At least three dose 
levels plus control 
At least 20 pregnant 
females per group 
Exposure for majority 
of normal lifespan 
At least three dose 
levels plus control 
At least 50 males and 
females per group 

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End points (general toxicity) 
As in OECD TG 416 / EU B.35 

As described in OECD TG 443 / EU B.56 

Clinical observations 
Body weight and food/water consumption 
Macroscopical examination of all dams for any 
structural abnormalities or pathological changes 
that may have influenced the pregnancy 

Clinical observations 
Body weight and food/water consumption 
Gross necropsy (adult animals, special attention 
to reproductive organs) 
Organ weights (all adult males: testes, 
epididymides) 
Histopathology (reproductive organs in at least 
control and high-dose groups) 

Clinical observations 
Body weight and food/water consumption 

Clinical observations (special attention to 
tumour development) 
Body weight and food consumption 
Gross necropsy 
Histopathology (all groups – all grossly visible 
tumours or lesions suspected of being tumours; 
at least control and high-dose groups – brain, 
pituitary, thyroid, parathyroid, thymus, lungs, 
heart, salivary glands, liver, spleen, kidneys, 
adrenals, oesophagus, stomach, duodenum, 
jejunum, ileum, caecum, colon, rectum, uterus, 
urinary bladder, lymph nodes, pancreas, 
gonads, accessory sex organs, female 
mammary gland, skin, musculature, peripheral 
nerve, spinal cord, sternum with bone marrow 
and femur, eyes) 

 
 
 
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3.3.3.  Specific system/organ toxicity 

3.3.3.1.  General aspects 

For some specific system/organ effects, the EU testing methods and the OECD guidelines may 
not provide for adequate characterisation of the toxicity. There may be indications of such effects 
in the standard studies for systemic toxicity or from SARs. For adequate characterisation of the 
toxicity and, hence, the risk to human health, it may be necessary to conduct studies using other 
established methods. 

Some specific investigation of organ/systemic toxicity (e.g. hepatotoxicity and nephrotoxicity) is 
undertaken  as  part  of  the  repeated  dose  toxicity  tests.  Reproductive  toxicity  is  specifically 
examined  using  appropriate  methods  (see  Section 3.6  of  this  guidance  document).  Specific 
investigation  (or  further  investigation)  of  any  organ/system  toxicity  (e.g.  kidney,  cardiac, 
adrenal, thyroid) may sometimes be considered necessary and should be addressed on a case-
by-case basis. 

3.3.3.2.  Neurotoxicity 

3.3.3.2.1. 

Definition of neurotoxicity 

Neurotoxicity is the induction by a chemical of adverse effects in the central or peripheral nervous 
system  or  in  the  sense  organs.  It  is  useful  for  the  purpose  of  hazard  and  risk  assessment  to 
differentiate sense-organ-specific effects from other effects that lie within the nervous system. 
A substance is considered neurotoxic if it induces a reproducible lesion in the nervous system or 
a reproducible pattern of neural dysfunction. 

3.3.3.2.2. 

Introduction 

It  is  recommended  that  a  hierarchical  approach  is  taken  in  the  investigation  of  the  potential 
neurotoxicity  of  substances.  The  starting  point  for  the  testing  strategy  should  be  exposure 
considerations, in  vitro  data and SARs, and  should proceed using data already available  from 
base  set  tests  to  more  specific  testing.  Thus,  any  indications  of  specific  or  non-specific 
neurotoxicity in the acute and repeated dose toxicity tests should be carefully noted. In addition, 
if there are already alerts from SARs or available information on the relevant chemical species 
or similar substances, and repeated dose toxicity is planned, it would be of benefit to investigate 
neurotoxicity  within  the  repeated  dose  test.  The  same  would  apply  for  consideration  of 
developmental neurotoxicity investigations within the reproductive toxicity generation tests. 

The  present  EU  and  OECD  28-day  and  90-day  oral  tests  (EU B.7  and  OECD  TG 407,  1995; 
EU B.26 and OECD TG 408, 1998) examine a number of simple nervous system end points (e.g. 
clinical observations of motor and autonomous nervous system activity, histopathology of nerve 
tissue), which should be regarded as the starting point for evaluation of a substance’s potential 
to cause neurotoxicity. It should be recognised that the standard 28- and 90-day tests measure 
only some aspects of nervous system structure and function, while other aspects, for example 
learning  and  memory  and  sensory  function,  are  not  or  only  superficially  tested.  SAR 
considerations may prompt the introduction of additional parameters to be tested in standard 
toxicity tests or the investigation of, for example, delayed neurotoxicity (EU B.37 and OECD TG 
418; EU B.38 and OECD TG 419). Any indication of the potential neurotoxicity of substances can 
also be a trigger for testing for developmental neurotoxicity (see also DWD Guidance Volume I). 

If there is no indication of neurotoxicity in humans, no indication in adequately performed acute 
and  repeated  dose  toxicity  tests  and  none  from  SARs,  it  is  not  necessary  to  conduct  any 
additional tests for neurotoxicity. 

 
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3.3.3.2.3. 

Structure–activity considerations 

Structural alerts are used only as a positive indication of neurotoxic potential. Substance classes 
with an alert for neurotoxicity may include organic solvents (for chronic toxic encephalopathy), 
organophosphorus  compounds  (for  delayed  neurotoxicity)  and  carbamates  (for  cholinergic 
effects). Several estimation techniques are available, one of which is the rule-based Derek Nexus 
system. The rule base comprises the following hazards and structural alerts: organophosphate 
(for  direct  and  indirect  anticholinesterase  activity),  N-methyl  or  N,N-dimethyl  carbamate  (for 
direct anticholinesterase activity) and gamma-diketones (for neurotoxicity). Another repository 
of  models  is  the  JRC’s  (Q)SAR  Model  Database  ((Q)SAR  model  reporting  framework 
inventory) ( 46), which is intended to provide information on (Q)SAR models submitted to the JRC 
for peer review. It should be noted that no formal adoption process for (Q)SAR models exists or 
is provided for under the DWD. The validity, applicability and adequacy of (Q)SAR models are 
assessed individually, with the prediction generated for the target chemical ( 47). 

3.3.3.2.4. 

Assessment of available information or results from initial 
testing 

Signs  of  neurotoxicity  in  standard  acute  or  repeated  dose  toxicity  tests  may  be  secondary  to 
other systemic toxicity or to discomfort from physical effects such as a distended or blocked GI 
tract. Nervous system effects seen at dose levels near or above those causing lethality should 
not be considered, in isolation, to be evidence of neurotoxicity. In acute toxicity studies where 
high doses are administered, clinical signs are often observed that are suggestive of effects on 
the  nervous  system  (e.g.  observations  of  lethargy,  postural  or  behavioural  changes),  and  a 
distinction should be made between specific and non-specific signs of neurotoxicity. 

Neurotoxicity may be indicated by the following signs: morphological (structural) changes in the 
central  or  peripheral  nervous  system  or  in  special  sense  organs,  neurophysiological  changes 
(e.g.  electroencephalographic  changes),  behavioural  (functional)  changes  or  neurochemical 
changes (e.g. neurotransmitter levels). 

The  type,  severity,  number  and  reversibility  of  the  effect  should  be  considered.  Generally,  a 
pattern  of  related  effects  is  more  persuasive  evidence  of  neurotoxicity  than  one  or  a  few 
unrelated effects. Available data should be assessed in a WoE to conclude whether an effect may 
be considered a concern for neurotoxicity. 

It  is  important  to  ascertain  whether  the  nervous  system  is  the  primary  target  organ.  The 
reversibility of neurotoxic effects should also be considered. Reversible effects may be of high 
concern depending on the severity and nature of the effects. In this context, it should be kept 
in mind that effects  observed in experimental animals that appear harmless might be  of high 
concern in humans depending on the setting in which they occur (e.g. sleepiness in itself may 
not  be  harmful,  but  in  relation  to  operating  machinery  it  is  an  effect  of  high  concern). 
Furthermore, the possibility that a permanent lesion has occurred cannot be excluded, even if 
the  overt  effect  is  transient.  The  nervous  system  possesses  reserve  capacity,  which  may 
compensate  for  the  damage,  but  the  resulting  reduction  in  the  reserve  capacity  should  be 
regarded  as  an  adverse  effect.  Compensation  may  be  suspected  if  a  neurotoxic  effect  slowly 
resolves  during  the  lifespan.  This  could  be  the  case  for  developmental  neurotoxicants  (see 
Section 3.6). Irreversible neurotoxic effects are of high concern and usually involve structural 
changes, although, at least in humans, lasting functional effects (e.g. depression, involuntary 

(46)  https://jeodpp.jrc.ec.europa.eu/ftp/jrc-opendata/EURL-ECVAM/datasets/QSARDB/LATEST/qsardb.html. 

(47)  https://echa.europa.eu/documents/10162/13655/pg_report_qsars_en.pdf/407dff11-aa4a-4eef-a1ce-

9300f8460099. 

 
 
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motor tremor) are suspected to occur as a result of neurotoxicant exposure, apparently without 
morphological abnormalities. 

3.3.3.2.5. 

Further neurotoxicity testing 

If  the  data  acquired  from  the  standard  systemic  toxicity  tests  are  inadequate  or  provide 
indications  of  neurotoxicity  that  are  not  sufficiently  investigated  for  risk  characterisation,  the 
nature  of  further  investigation  will  need  to  be  considered.  Additional  guidance  is  provided  in 
DWD  Guidance  Volume I  and  in  ECHA’s  Guidance  on  the  Biocidal  Products  Regulation – 
Volume III: Human health – Part A: Information requirements ( 48). 

3.3.3.3.  Immunotoxicity 

3.3.3.3.1. 

Definition of immunotoxicity 

Immunotoxicity  is  defined  as  any  adverse  effect  on  the  immune  system  that  can  result  from 
exposure to a range of environmental agents, including chemicals (IPCS, 2012). 

An immunotoxic response may occur when the immune system is the target of a chemical insult; 
this in turn can result in either immunosuppression and a subsequent decreased resistance to 
infection and certain forms of neoplasia, or immune dysregulation, which exacerbates allergy or 
autoimmunity reaction. Alternatively, toxicity may arise when the immune system responds to 
an  antigenic  specificity  of  the  chemical  as  part  of  a  specific  immune  response  (i.e.  allergy  or 
autoimmunity).  Changes  of  immunological  parameters  may  also  be  a  secondary  response  to 
stress resulting from effects on other organ systems. Therefore, it must be recognised that in 
principle all chemical substances may be able to influence parameters of the immune system if 
administered at a sufficiently high dosage; however, these may not constitute substance-specific 
effects.  An  immunotoxic  effect  should  not  be  disregarded  until  an  adequate  investigation  has 
been performed. 

The  IPCS  published  Guidance  for  Immunotoxicity  Risk  Assessment  for  Chemicals  (IPCS 
Harmonization  Project  Document  No 10),  focused  on  immunosuppression,  inadvertent 
immunostimulation  and  autoimmunity  caused  by  chemical  exposure ( 49).  Therefore,  the  IPCS 
Guidance  for  Immunotoxicity  Risk  Assessment  for  Chemicals  (IPCS,  2012)  must  be  consulted 
together  with  this  volume  of  the  DWD  guidance  when  performing  the  assessment  of  this  end 
point. 

3.3.3.3.2. 

Introduction 

The  toxicological  significance  of  immune  responses  is  currently  under  discussion  by  several 
scientific groups (e.g. European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), 
IPCS).  Immunotoxicity  is  of  particular  concern  for  test  substances  that  induce  toxicity  on  the 
immune  system  at  dose  levels  below  those  that  induce  toxicity  at  other  target  sites.  If  the 
immunotoxicity  is  the  critical  effect,  it  is  recommended  to  assess  immune  effects  in  the  risk 
assessment  process  as  for  any  other  toxic  effect  (IPCS,  1996).  As  the  revised  test  methods 
(OECD TG 407 and 408) begin to be applied routinely, it is expected that the database on the 
immunotoxic  potential  of  substances  will  grow  and  experience  on  the  evaluation  of  immune 

(48)  https://echa.europa.eu/documents/10162/2324906/bpr_guidance_vol_iii_part_a_en.pdf/05e4944d-106e-9305-

21ba-f9a3a9845f93?t=1648525287369. 

(49)  https://iris.who.int/bitstream/handle/10665/330098/9789241503303-eng.pdf?sequence=1. 

 
 
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effects  will  increase.  The  test  guidelines  are  intended  primarily  as  a  screening  for 
immunotoxicity, and, depending on the results, immediate further testing may be needed. 

3.3.3.3.3. 

Immunosuppression 

In vivo animal studies. The basis of the currently recommended approach to assessment of 
the potential immunotoxicity of a new substance is that many immunotoxic substances can be 
identified  in  the  context  of  more  general  toxicity  testing,  such  as  the  28-day  repeated  dose 
toxicity study (OECD TG 407), the 90-day repeated dose toxicity study (OECD TG 408) and the 
extended  one-generation  reproduction  toxicity  study  (OECD  TG 443).  Special  studies  to 
characterise effects of concern for immunotoxicity are used only when necessary for adequate 
risk characterisation. The nature of special studies and when they should be conducted need to be 
decided on a case-by-case basis. In particular, the use of in vivo tests should not be undertaken 
without detailed consideration of the need for such studies (IPCS, 2012). 

The protocols of both the EU and the OECD TG 407 (28-day) and OECD TG 408 (90-day) studies 
include the measurement of thymus and spleen weights and histopathological examination of 
certain lymphoid tissues (i.e. thymus, draining and distant lymph nodes, Peyer’s patches, bone 
marrow  section)  in  addition  to  the  total  and  differential  white  blood  cell  counts  and  spleen 
histopathology  required  by  the  previous  EU  method.  These  tissues  all  have  immunological 
functions, and changes to them can be indicative of adverse effects on the immune system. 

Indications of immunotoxicity from standard repeated dose toxicity studies may include one or 
more of the following signs: 

•  morphological changes of lymphoid organs and tissues, including bone marrow (e.g. altered 
cellularity/size of major compartments), in the absence of other relevant substance-specific 
effects that cause this change; 

•  weight  changes  of  lymphoid  organs  in  the  absence  of  other  relevant  substance-specific 

effects that cause this change; 

• 

• 

changes in haematology parameters (e.g. white blood cell number, differential cell counts of 
lymphocytic, monocytic and granulocytic cells); 

changes  in  clinical  chemistry  parameters  (e.g.  serum  protein  levels,  immunoglobulin 
concentrations if determined). 

It has to be noted that the effects listed above may not be sufficient by themselves to come to 
a conclusion on immunotoxicity but should rather be considered in a WoE approach, taking into 
account all the available information. 

If there are no indications of immunotoxicity in the 28-day (or 90-day) toxicity test and none 
from SARs, no further specific investigation for immunotoxicity is normally required. 

However, the abovementioned effects may also raise concern for mechanisms/modes of action 
associated with developmental immunotoxicity. For regulatory purposes, such concern is further 
investigated in cohort 3 of the extended one-generation reproductive toxicity study via the T-
cell-dependent antibody response assay, which is a so-called functional assay. However, it has 
to be considered that, as the concern for justifying the request for developmental immunotoxicity 
study  is  based  on  data  generated  in  adult  animals,  it  is  possible  to  miss  substances  causing 
immunomodulation in developing animals. 

Further testing to investigate immune function (e.g. a T-cell function  test for  substances that 
cause  histopathological  changes  in  the  thymus,  host  resistance  models)  should  be  conducted 

 
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only if the results of such studies can be interpreted in relation to the risk assessment for the 
relevant  chemical  species.  In  many  cases,  the  observation  of  the  morphological  changes  or 
changes in haematology and clinical chemistry parameters, together with a NOAEL/LOAEL/BMD 
for  those  changes,  will  be  sufficient  for  screening.  Functional  assays  may  provide  valuable 
information for identifying immunotoxic effects and, in some cases, can be more sensitive than 
non-functional assays. However, it should be noted that the observation of the immunological 
changes  discussed  above  may  not  necessarily  reflect  a  direct  immunotoxic  effect  but  may  be 
secondary to other effects. 

For immunotoxicity testing, a list of reviews of principles and methods from the IPCS is provided 
in  its  guidance  on  information  requirements  (IPCS,  2012),  along  with  a  list  of  available  test 
methods, including assays for the assessment of autoimmunity. 

Non-animal methods 

Depending on the mode of action suspected and the effects observed, in vitro studies may be 
able to provide useful mechanistic insight for the assessment of immunotoxicity. To assess the 
immunotoxicity of chemicals, a huge number of non-animal testing models have been published; 
however,  only  a  few  cell-based  test  methods  have  reached  the  stage  of  validation  and 
acceptance  by  international  regulatory  bodies,  such  as  OECD  TG 444A  (IL-2  Luc  assay). 
Therefore, the majority of reported tests still fall into the category of non-guideline methods; 
however, the results obtained from these tests may nevertheless be used to support regulatory 
decisions. Following the developments in the field of immunotoxicity is recommended, as new 
methods and approaches to assess immunotoxicity may become available. 

A detailed review paper on non-animal approaches that could be used to test chemicals for their 
potential immunotoxic effects has been published under the Inter-Organization Programme for 
the Sound Management of Chemicals50. The detailed review paper aims to present and discuss 
the  application  and  interpretation  of  in  vitro  immunotoxicity  assays,  covering  mainly 
immunosuppression, and to define an in vitro tiered approach to testing and assessment. 

Currently,  there  are  some  initiatives  by  the  Johns  Hopkins  Center  for  Alternatives  to  Animal 
Testing to establish a framework to foster the refinement and development of new alternative 
test methods suitable for screening of developmental immunotoxic compounds (51). 

Despite the need for an in vitro tiered system to evaluate immunotoxicity, there is currently no 
combination  of  OECD  test  guidelines  that  can  be  used  to  conclude  on  immunosuppression  in 
vitro. It is clear that one assay alone will not be able to cover all of the potential adverse effects 
of chemicals on the immune system and that a larger set of assays that will cover the spectrum 
of immunotoxicity is needed. 

A tiered testing strategy is proposed to assess immunotoxicity in vitro (Gennari et al., 2005). In 
the  proposed  tiered  approach,  pre-screening  for  direct  immunotoxicity  in  vitro  begins  by 
evaluating myelotoxicity (tier 1). Compounds capable of damaging or destroying bone marrow 
cells will most likely have immunotoxic effects, as the majority of immune cells are derived from 
a common precursor located in adult bone marrow. If compounds are not potentially myelotoxic, 
they should be tested for direct leukotoxicity, defined as toxicity to any cell of the lymphoid or 
myeloid lineages (tier 2). Compounds should then be tested for immunotoxicity using various 
approaches, such as T-cell-dependent antibody response, lymphocyte proliferation assay, mixed 
leucocyte reaction, natural killer cell assay, dendritic cell maturation assay, human whole-blood 
cytokine release assay (HWBCRA) and fluorescent cell chip assay (tier 3). 

(50)  ENV/CBC/MONO(2022)16 (https://one.oecd.org/document/ENV/CBC/MONO(2022)16/En/pdf). 

(51)  https://caat.jhsph.edu/developmental-immunotoxicity/. 

 
 
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Among these assays, the HWBCRA has the advantage of comprising multiple cell types in their 
natural proportion and environment, allowing the evaluation of both monocyte and lymphocyte 
functions by using selective stimuli (Langezaal et al., 2001, 2002), while omics techniques can 
provide  additional  mechanistic  understanding  and  hold  promise  for  the  characterisation  of 
classes of compounds and prediction of specific toxic effects (Hochstenbach et al., 2010; Shao 
et al., 2014; Schmeits  et al., 2015). The IL-2  Luc assay also allows high-throughput analysis 
(Kimura et al., 2018), which will greatly expand the opportunities for in vitro testing. The colony 
forming unit – granulocyte/macrophage assay, the HWBCRA as a pyrogen test and the IL-2 Luc 
assay have undergone validation for reproducibility and predictive capacity. 

It  is  likely  that  multiple  assays  will  be  required  to  define  immunotoxicants  because  of  the 
complexity  and  varied  components  of  the  immune  system  (e.g.  innate  or  adaptive  immune 
responses). For example, for a combination of in vitro assays that may be used to predict the 
immunotoxicity of chemicals, please refer to the detailed review paper (52). 

3.3.3.4.  Effects on the endocrine system 

The  endocrine  system  consists  of  a  set  of  glands  that  includes  the  thyroid,  gonads  and  the 
adrenal glands, and the hormones they produce, such as thyroxine, oestrogen, testosterone and 
adrenaline, which help guide the development, growth, reproduction and behaviour of animals, 
including human beings. 

to 

the  CLP 

(EC)  No 1272/2008 (53)),  Annex 1, 
(Regulation 
According 
Section 3.11.1.1(a), ‘“endocrine disruptor” means a substance or a mixture that alters one or 
more functions  of the  endocrine system and consequently  causes adverse  effects in an intact 
organism, its progeny, populations or subpopulations’. 

regulation 

EDs are believed to interfere with the endocrine system, leading to adverse effects by one or 
more modes of action, depending on the individual substance. There are three possible general 
modes of action, as listed below: 

•  by mimicking the action of a naturally produced hormone such as oestrogen or testosterone, 

thereby setting off similar chemical reactions in the body; 

•  by  blocking  the  receptors  in  cells  receiving  the  hormones  (hormone  receptors),  thereby 

preventing the action of normal hormones; 

•  by affecting the synthesis, transport, metabolism and excretion of hormones, thus altering 

the concentration of normal hormones. 

In relation to hazard identification, the procedures described in the ECHA/EFSA guidance on the 
identification of EDs in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009 (54) 
should be considered. 

3.3.3.5.  Remaining uncertainty 

The following elements contribute to the uncertainty in the determination of a threshold for the 
critical effects and the selection of the AF (see also Section 4.2.2.2). 

(52)  https://one.oecd.org/document/ENV/CBC/MONO(2022)16/En/pdf. 

(53)  https://eur-lex.europa.eu/eli/reg/2008/1272/oj. 

(54)  https://echa.europa.eu/fi/-/guidance-on-identifying-endocrine-disruptors-published. 

 
 
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3.3.3.5.1. 

Threshold of the critical effect 

In the determination of the overall threshold for repeated dose toxicity, all relevant information 
is evaluated to determine the lowest dose that induces an adverse effect (i.e. LOAEL or lowest 
observed  adverse  effect  concentration  (LOAEC))  and  the  highest  level  with  no  biologically  or 
statically significant adverse effects (i.e. NOAEL or NOAEC). In this assessment, all toxicological 
responses  are  taken  into  account  and  the  critical  effect  is  identified.  The  uncertainty  in  the 
threshold depends on the strength of the data and is largely determined by the design of the 
underlying  experimental  data.  Parameters  such  as  group  size,  study  type/duration  and  the 
methodology need to be taken into account in the assessment of the uncertainty in the threshold 
of the critical effect(s). 

The NOAEL is typically used as the starting point for the derivation of the threshold level (e.g. 
DNEL). In cases  where  a NOAEL has not been  achieved, a  LOAEL may be used, provided the 
available information is sufficient for a robust hazard assessment. The BMD may also be used as 
the starting point. 

The selection of the NOAEL or LOAEL is usually based on the dose levels used in the most relevant 
toxicity  study,  without  considering  the  shape  of  the  dose–response  curve.  Therefore,  the 
NOAEL/LOAEL may not reflect the true threshold for the adverse effect. In contrast, the BMD is 
a  statistical  approach  for  the  determination  of  the  threshold  and  relies  on  the  dose–response 
curve.  Alternatively,  mathematical  curve-fitting  techniques  or  statistical  approaches  exist  for 
determining  the  threshold  for  an  adverse  effect.  The  use  of  such  approaches  (e.g.  BMD)  to 
estimate the threshold should be considered on a case-by-case basis and are usually used for 
higher-tier hazard characterisation refinement. 

3.3.3.5.2. 

Other considerations 

Another situation may arise when testing is not technically possible, a waiving option indicated 
in Part 2, Section 2 of Annex V to Commission Implementing Decision (EU) 2024/365. In such 
cases, approaches such as (Q)SAR, category formation and read-across may be helpful in the 
hazard characterisation; they should also be considered for information that might be suitable 
as a surrogate for a dose descriptor. Alternatively, generic threshold approaches, for example 
the  threshold  of  toxicological  concern,  might  be  considered  for  the  starting  point  of  a  risk 
characterisation as risk management tools to estimate negligible exposure potential (for more 
information,  see  Appendix 1-4  of  Guidance  on  the  Biocidal  Products  Regulation –  Volume III 
human health – Assessment & evaluation (Parts B+C) (55)). 

3.3.4.  Conclusions on repeated dose toxicity 

Potentially relevant studies should be judged for quality, and studies of high quality should be 
given  more  weight  than  those  of  lower  quality.  When  both  epidemiological  and  experimental 
data are available, similarity of effects between humans and animals is given more weight. If 
the  mechanism  or  mode  of  action  is  well  characterised,  this  information  is  used  in  the 
interpretation of observed effects in either human or animal studies. WoE is not to be interpreted 
as simply tallying the number of positive and negative studies, nor does it imply an averaging 
of the doses or exposures identified in individual studies that may be suitable as starting points 
for risk assessment. The study or studies used for the starting point are identified by an informed 
and expert evaluation of all the available evidence. Recommendations on how to perform a WoE 

(55)

https://echa.europa.eu/documents/10162/2324906/biocides_guidance_human_health_ra_iii_part_bc_en.pdf/

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are provided in Section 3.1.4 of this guidance, as well as in the EFSA guidance on the use of the 
WoE approach (56) and in Chapter R.4 of the ECHA guidance (57). 

The available repeated dose toxicity data should be evaluated in detail for a characterisation of 
the health hazards upon repeated exposure. In this process, an assessment of all toxicological 
effect(s), their dose–response relationships and possible thresholds are taken into account. The 
evaluation should include an assessment of the severity of the effect, its adversity, or if it is a 
precursor  to  a  more  significant  effect  or  secondary  to  general  toxicity.  Correlations  between 
changes  in  several  parameters,  for  example  between  clinical  or  biochemical  measurements, 
organ weights and (histo)pathological effects, will be helpful in the evaluation of the nature of 
the effects. Further guidance on this issue can be found in the publications of ECETOC (2021) ( 58) 
and Inchem (2020) ( 59). 

The effects data are also analysed for indications of potential serious toxicity of target organs or 
specific  organ  systems  (e.g.  neurotoxicity  or  immunotoxicity),  delayed  effects  or  cumulative 
toxicity. Furthermore, the evaluation should take into account the study details and determine 
if  the  exposure  conditions  and  duration  and  the  parameters  studied  are  appropriate  for  an 
adequate characterisation of the toxicological effect(s). 

If  an  evaluation  allows  the  conclusion  that  the  information  of  the  repeated  dose  toxicity  is 
adequate for a robust characterisation of the toxicological hazards, including an estimate of a 
dose descriptor (BMD/NOAEL/LOAEL), and the data are adequate for risk assessment under the 
DWD, no further testing will be necessary unless there are indications of further risk. 

Another consideration is whether the study duration was appropriate for an adequate expression 
of the toxicological effects. If the critical effect involves serious specific system or target organ 
toxicity  (e.g.  haemolytic  anaemia,  neurotoxicity  or  immunotoxicity),  delayed  effects  or 
cumulative toxicity, and a threshold has not been established, dose extrapolation may not be 
appropriate and further studies are required. In this case a specialised study is likely to be more 
appropriate  for  an  improved  hazard  characterisation  and  should  be  considered  instead  of  a 
standard short-term rodent or subchronic toxicity test at this stage. 

3.3.5.  Concluding on suitability for risk assessment 

It is necessary to identify the so-called dose descriptor, that is, an appropriate threshold dose 
for  the  critical  effect  as  the  starting  point  for  DNEL  derivation,  namely  a  BMD  or  NOAEL.  If  a 
NOAEL cannot be identified, the LOAEL may be used instead, provided the data are adequate for 
a robust hazard assessment. 

It must be noted that the dose descriptor should be route specific. As explained in Section 5, the 
oral route is the relevant one for relevant species migrated in the drinking water. Thus, in cases 
when  only  animal  data  with  dermal  or  inhalation  exposure  are  available,  and  humans  are 
exposed via ingestions, as is the case for drinking water, a threshold level for the oral route is 
needed,  that  is,  route-to-route  extrapolation  is  needed.  Guidance  on  this  route-to-route 
extrapolation is provided in Chapter 4 ‘Hazard assessment’. 

(56)  https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2017.4971. 

(57)  https://echa.europa.eu/documents/10162/17235/information_requirements_r4_en.pdf/d6395ad2-1596-4708-

ba86-0136686d205e?t=1323782558175. 

(58)  https://www.ecetoc.org/wp-content/uploads/2021/10/ECETOC-TR-138-Guidance-on-Dose-Selection_Final.pdf. 

(59)  https://cdn.who.int/media/docs/default-source/food-safety/publications/chapter5-dose-response.pdf?sf. 

 
 
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3.4.  Mutagenicity 

67 

The section on mutagenicity in DWD Guidance Volume I should be considered together with the 
elements described in this section for the assessment of mutagenicity. 

Section 1  of  Annex VI  to  Commission  Implementing  Decision  (EU)  2024/365  lists  the  criteria 
when no hazard assessment needs to be performed. 

3.4.1.  Definition 

Mutagenicity  refers  to  the  induction  of  permanent  transmissible  changes  in  the  amount  or 
structure of the genetic material of cells or organisms. These changes may involve a single gene 
or gene segment, a block of genes or chromosomes. Several mutagenicity end points (i.e. gene 
mutation, clastogenicity and aneugenicity) can be distinguished. The term gene mutation refers 
to permanent changes in the base sequences of a certain gene. The term clastogenicity is used 
for  agents  giving  rise  to  structural  chromosome  aberrations.  A  clastogen  can  cause  breaks  in 
chromosomes that result in the loss or rearrangement of chromosome segments. Aneugenicity 
(aneuploidy induction) refers to the effects of agents that give rise to a change (gain or loss) in 
chromosome number in cells. An aneugen can cause loss or gain of chromosomes, resulting in 
cells that do not have an exact multiple of the haploid number. For example, three chromosomes 
of the same number, or trisomy (e.g. trisomy 21, characteristic of Down’s syndrome), is a form 
of aneuploidy. 

Genotoxicity  is  a  broader  term  that  refers  to  processes  that  alter  the  structure,  information 
content or segregation of DNA and are not necessarily associated with mutagenicity. Thus, tests 
for genotoxicity include tests that provide an indication of induced damage to DNA (but not direct 
evidence of mutation) via effects such as DNA strand breaks, unscheduled DNA synthesis, sister 
chromatid  exchange  and  DNA  adduct  formation  or  mitotic  recombination,  as  well  as  tests  for 
mutagenicity. 

The  chemical  and  structural  complexity  of  the  chromosomal  DNA  and  associated  proteins  of 
mammalian cells, and the multiplicity of ways in which changes to the genetic material can be 
affected make it difficult to give more precise, discrete definitions. 

In the risk assessment of the relevant chemical species, it is necessary to address the potential 
effect of mutagenicity. It can be expected that some of the available data will have been derived 
from  tests  conducted  to  investigate  potentially  harmful  effects  on  genetic  material 
(genotoxicity). Hence, both the terms mutagenicity and genotoxicity are used in this document. 

The  aim  of  testing  for  genotoxicity  is  to  assess  the  potential  of  relevant  chemical  species  to 
induce genotoxic effects that may lead to cancer or cause mutagenicity in humans. Genotoxicity 
data are also used in risk characterisation of substances. 

Alterations to the genetic material of cells may occur spontaneously or be induced as a result of 
exposure  to  ionising  or  ultraviolet  radiation,  or  to  genotoxic  substances.  In  principle,  human 
exposure  to  substances  that  are  mutagens  may  result  in  increased  frequencies  of  mutations 
above the background level. 

Mutations in somatic cells may be lethal or may be transferred to daughter cells with deleterious 
consequences  for  the  affected  organism  (e.g.  cancer  may  result  when  they  occur  in  proto-
oncogenes,  tumour  suppressor  genes  and/or  DNA  repair  genes),  ranging  from  trivial  to 
detrimental or lethal. 

There is considerable evidence of a positive correlation between the mutagenicity of substances 
in vivo and their carcinogenicity in long-term studies with animals. Genotoxic carcinogens are 

 
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chemicals for which the most plausible mechanism of carcinogenic action involves genotoxicity 
(Kang et al., 2013). 

Heritable  damage  to  the  offspring,  and  possibly  to  subsequent  generations,  may  follow  after 
parents are exposed to substances that are mutagens if mutations are induced in parental germ 
cells.  To  date,  all  known  germ  cell  mutagens  are  also  mutagenic  in  somatic  cells  in  vivo. 
Substances that are mutagenic in somatic cells may produce heritable effects if they, or their 
active metabolites, reach the genetic material of germ cells. Conversely, substances that do not 
induce mutations in somatic cells in vivo would not be expected to be germ cell mutagens. 

3.4.2.  Data to be used in the assessment of genotoxicity/mutagenicity 

Genotoxicity is a complex end point and requires evaluation by expert judgement. For both steps 
of  the  effects  assessment,  namely  hazard  identification  and  dose  (concentration)–response 
(effect) assessment, it is very important to evaluate the data with regard to their adequacy and 
completeness. The evaluation of adequacy must address the reliability and relevance of the data 
in  a  way  outlined  in  the  introductory  section.  The  completeness  of  the  data  refers  to  the 
conclusion drawn on whether the available information is adequate and fulfils the requirements 
under  Section 2  of  Annex V  to  Commission  Implementing  Decision  (EU)  2024/365.  Such  a 
conclusion  relies  on  WoE  approaches,  which  categorise  available  information  based  on  the 
methods  used:  guideline  tests,  non-guideline  tests  and  other  types  of  information  that  may 
justify  adaptation  of  the  standard  testing  regime.  Such  a  WoE  approach  also  includes  an 
evaluation of the available data as a whole, that is, both over and across toxicological end points. 

This approach provides a basis for deciding whether further information is needed on end points 
for  which  specific  data  appear  inadequate  or  not  available,  or  whether  the  requirements  are 
fulfilled. 

3.4.2.1.  Non-human data for mutagenicity 

3.4.2.1.1. 

Non-testing data for mutagenicity 

See Section 3.1.2.2.5 (on (Q)SARs) and Section 3.1.3 (on the read-across approach). 

3.4.2.1.2. 

Testing data for mutagenicity 

Test methods preferred for use are listed in Tables 7–9. Some of these have officially adopted 
EU/OECD guidelines for genotoxicity testing. 

 
 
 
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In vitro data 

Table 7. In vitro test methods 

Test 

Bacterial reverse 
mutation test 

Genotoxic end points 
measured  
Gene mutations  

Gene mutations  

Gene mutations and 
structural chromosome 
aberrations  

In vitro 
mammalian cell 
gene mutation 
tests using the 
Hprt and Xprt 
genes 

In vitro 
mammalian cell 
gene mutation 
tests using the 
thymidine kinase 
(TK) gene 

In vitro 
mammalian 
chromosome 
aberration test 

Structural (and 
numerical) chromosome 
aberrations 

In vitro 
mammalian 
micronucleus test 

Structural and numerical 
chromosome aberrations 

69 

Principle of the test method 

EU/OECD guideline 

The test uses amino acid requiring 
strains of bacteria to detect 
(reverse) gene mutations (point 
mutations and frameshifts). 

The test identifies chemicals that 
induce gene mutations in the Hprt 
and Xprt genes of established cell 
lines. 

EU B.13/14 / OECD 
TG 471 

EU B.17 / OECD 
TG 476 

EU B.67 / OECD 
TG 490 

EU B.10 / OECD 
TG 473 

EU B.49 / OECD 
TG 487 

The test identifies chemicals that 
induce gene mutations in the TK 
gene of the L5178Y mouse 
lymphoma or TK6 human 
lymphoblastoid cell line. If colonies 
in a TK mutation test are scored 
using the criteria of normal growth 
(large / early appearing) and slow 
growth (small/late-appearing) 
colonies, gross structural 
chromosome aberrations may be 
measured, since mutant cells that 
have suffered the most extensive 
genetic damage have prolonged 
doubling times and are more likely 
to form small/late-appearing 
colonies. 

The test identifies chemicals that 
induce chromosome aberrations in 
cultured mammalian established 
cell lines, cell strains or primary 
cell cultures. An increase in 
polyploidy may indicate that a 
chemical has the potential to 
induce numerical chromosome 
aberrations. However, this test is 
not designed to measure 
aneuploidy. 

The test identifies chemicals that 
induce micronuclei in the 
cytoplasm of interphase cells. 
These micronuclei may originate 
from acentric fragments or whole 
chromosomes, and the test thus 
has the potential to detect both 
clastogenic and aneugenic 
chemicals. 

 
 
 
 
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Animal data 

Table 8. Somatic cells – in vivo test methods 

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Test 

In vivo mammalian 
bone marrow 
chromosome 
aberration test 

Genotoxic end 
points measured  
Structural (and 
numerical) 
chromosome 
aberrations 

In vivo mammalian 
erythrocyte 
micronucleus test 

Structural and 
numerical 
chromosome 
aberrations 

Unscheduled DNA 
synthesis test with 
mammalian liver 
cells in vivo (a) 

DNA repair  

Transgenic rodent 
somatic and germ 
cell gene mutation 
assay 

Gene mutations /  

In vivo mammalian 
alkaline comet assay 

DNA strand breaks in 
eukaryotic cells 

Mammalian 
erythrocyte Pig-a 
gene mutation assay 

Gene mutations 

EU/OECD 
guideline 

EU B.11 / OECD 
TG 475 

EU B.12 / OECD 
TG 474 

EU B.39 / OECD 
TG 486 

EU B.58 (b) / 
OECD TG 488 

EU B.62 / OECD 
TG 489 

OECD TG 470 

Principle of the test method 

The test identifies chemicals that 
induce structural chromosome 
aberrations in the bone marrow cells of 
animals, usually rodents. An increase 
in polyploidy may indicate that a 
chemical has the potential to induce 
numerical chromosome aberrations. 

The test identifies chemicals that cause 
micronuclei in erythroblasts sampled 
from bone marrow and/or peripheral 
blood cells of animals, usually rodents. 
These micronuclei may originate from 
acentric fragments or whole 
chromosomes, and the test thus has 
the potential to detect both clastogenic 
and aneugenic chemicals. 

The test identifies chemicals that 
induce DNA repair (measured as 
unscheduled ‘DNA’ synthesis) in liver 
cells of animals, commonly rats. The 
test is usually based on the 
incorporation of tritium labelled 
thymidine into the DNA by repair 
synthesis after excision and removal of 
a stretch of DNA containing a region of 
damage. 

The test can measure gene mutations 
in any tissue of an animal and may, 
therefore, also be used in specific site 
of contact tissues. 
The test can measure DNA strand 
breaks in any tissue of an animal and 
may, therefore, also be used in specific 
site of contact tissues. 

The erythrocyte Pig-a assay uses an 
endogenous mammalian gene, the 
phosphatidylinositol glycan class A gene 
(Pig-a), as a reporter of somatic cell 
gene mutations in erythroid precursor 
cells, primarily found in the bone 
marrow. The test can identify 
substances that cause gene mutations 
in these precursor cells, which are 
reflected in erythrocytes sampled from 
peripheral blood cells of animals, 
usually rodents. The test can be 
conducted without euthanising the 
animals, which facilitates integration of 
the Pig-a assay into many in vivo 
rodent testing protocols. 

 
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(a) 
This  test  is  no  longer  considered  appropriate;  therefore,  data  from  existing  tests  may  be  used  in  the  risk 
assessment,  but  no  new  tests  should  be  carried  out  for  the  purpose  of  Commission  Implementing  Decision (EU) 
2024/365 (see Commission Regulation 2023/464 of 3.3.2023 amending, for the purpose of its adaptation to technical 
progress,  the  Annex  to  Regulation  (EC)  No 440/2008  laying  down  test  methods  pursuant  to  Regulation  (EC) 
No 1907/2006 of the European Parliament and of the Council on the registration, evaluation, authorisation and restriction 
of  chemicals  (C(2023)1099 final),  https://environment.ec.europa.eu/publications/commission-regulation-amending-
purpose-its-adaptation-technical-progress-annex-regulation-ec-no_en). 

(b) 

Does not include the latest update to OECD TG 488. 

Table 9. Germ cells – in vivo test methods 

Test 

Mammalian 
spermatogonial 
chromosome 
aberration test 

Genotoxic end points 
measured 
Structural (and numerical) 
chromosome aberrations 

Rodent 
dominant 
lethal test (a) 

Structural and numerical 
chromosome aberration 

Gene mutation 

Transgenic 
rodent somatic 
and germ cell 
gene mutation 
assay 

Principle of the test method 

The test measures structural 
chromosome aberrations in 
mammalian, usually rodent, 
spermatogonial cells and is, therefore, 
expected to be predictive of induction 
of heritable mutations in germ cells. An 
increase in polyploidy may indicate 
that a chemical has the potential to 
induce numerical chromosome 
aberrations. This test is not designed 
to measure numerical abnormalities. 

The test measures dominant lethal 
effects causing embryonic or fetal 
death resulting from inherited 
dominant lethal mutations induced in 
germ cells of an exposed parent, 
usually the male. It is generally 
accepted that dominant lethals are due 
to structural and numerical 
chromosome aberrations. Rats or mice 
are recommended as the test species. 

The test measures gene mutations in 
spermatocytes of an animal and may, 
therefore, be used to obtain 
information about the mutagenic 
activity of a chemical in germ cells. 

EU/OECD 
guideline 

EU B.23 / 
OECD TG 483 

EU B.22 / 
OECD TG 478 

EU B.58 (b) / 
OECD TG 488 

(a) 
Data from existing tests may be used in the risk assessment; however, due to the known limitations of the test 
(e.g. high number of animals), no new tests should be carried out for the purpose of Commission Implementing Decision 
(EU) 2024/365. 

(b) 

Does not include the latest update to OECD TG 488. 

It  should  be  noted  that,  although  at  present  the  in  vivo  mammalian  alkaline  comet  assay  as 
described  in  OECD  TG 489  is  not  considered  appropriate  for  measuring  DNA  strand  breaks  in 
mature  germ  cells,  in  some  cases  available  in  vivo  comet  data  may  include  analysis  of  male 
gonadal cells from the seminiferous tubules. If such results are available, they may be relevant 
for the overall assessment of germ cell mutagenicity. 

Evaluation of genotoxicity test data should be carried out with care. 

Each  test  guideline  contains  criteria  for  the  acceptability  of  the  study  based  on  important 
parameters related to the study design and test conditions (e.g. acceptable cell type or animal 
species, number of cells used and scored or animals tested per group, dose/concentration levels 
and  the  number  of  test  doses/concentrations,  recommended  negative  and  positive  controls, 

 
 
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treatment schedule, exposure and sampling time(s), acceptable levels of (cyto)toxicity, evidence 
of target tissue exposure, laboratory proficiency demonstration) and criteria for the evaluation 
and interpretation of results (definition of clearly positive and clearly negative responses based 
on,  for  example,  statistical  analysis  or  threshold  values,  or  comparison  with  historical  control 
ranges for the negative and positive controls). 

In addition, further aspects described below need to be considered to determine the validity of 
study results. 

Regarding positive findings, responses generated only at highly toxic/cytotoxic concentrations 
should be interpreted with caution, and the presence or absence of a dose–response relationship 
should be considered. 

Particular points to take into account when evaluating negative test results include the following. 

•  The doses or concentrations of the test substance used (were they high enough?). 

•  Was the test system used sensitive to the nature of the genotoxic changes that might have 
been expected? For example, some in vitro test systems will be sensitive to point mutations 
and small deletions but not to mutagenic events that create large deletions. 

•  The  volatility  of  the  test  substance  (were  concentrations  maintained  in  tests  conducted  in 

vitro?). 

•  For studies in vitro, the possibility of metabolism not being active in the system, including 

those in extrahepatic organs. 

•  Was the test substance taken up by the test system used for in vitro studies? 

•  Was  a  sufficient  number  of  cells  scored/sampled  for  studies  in  vitro?  Has  the  appropriate 
number of samples / technical replicates been scored to support statistical significance of the 
putative negative result? 

•  For  studies  in  vivo,  was/were  the  most  appropriate  tissue(s)  sampled?  Did  the  substance 
reach the target organ? Or was the substance expected to act only at the site of contact due 
to  its  high  reactivity  or  insufficient  systemic  availability  (also  taking  TK  data  into 
consideration; for example, rate of hydrolysis and electrophilicity may be factors that need 
to be considered)? 

•  For studies in vivo, was sampling appropriate?  (Was a sufficient number  of animals used? 
Were sufficient sampling times used? Was a sufficient number of cells scored/sampled?) 

Different  results  between  different  test  systems  should  be  evaluated  with  respect  to  their 
individual significance. Examples of points to be considered are as follows. 

•  Different results obtained in non-mammalian systems and in mammalian cell tests may be 
addressed  by  considering  possible  differences  in  substance  uptake,  metabolism  or  the 
organisation of genetic material. Although the results of mammalian tests may be considered 
of higher significance, additional data may be needed to resolve contradictions. 

• 

If  the  results  of  indicator  tests  detecting  putative  DNA  lesions  (e.g.  DNA  binding,  DNA 
damage, DNA repair, sister chromatid exchange) are not in agreement with results obtained 
in  tests  for  mutagenicity,  the  results  of  mutagenicity  tests  are  generally  of  higher 
significance,  provided  that  appropriate  mutagenicity  tests  have  been  conducted.  This  is 
subject to expert judgement. 

 
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• 

If different findings are obtained in vitro and in vivo, in general, the results of in vivo tests 
indicate a higher degree of reliability. However, for evaluation of negative results in vivo, it 
should be considered whether the most appropriate tissues were sampled and whether there 
is  adequate  evidence  of  target  tissue  exposure.  The  assessment  of  contradicting  data  of 
comparable  quality  assessing  similar  end  points  should  be  done  on  a  case-by-case  basis, 
while considering the limitations of the respective source of the data. 

•  The  sensitivity  and  specificity  of  different  test  systems  may  vary  for  different  classes  of 
substances. If available testing data for other related substances permits assessment of the 
performance of difference assays for the class of substance under evaluation, the result from 
the test system known to produce more accurate responses would be given higher priority. 

Different  results  may  also  be  also  available  from  the  same  test  performed  by  different 
laboratories or on different occasions. In this case, expert judgement should be used to evaluate 
the data and to reach an overall conclusion. In particular, the quality of each of the studies and 
of  the  data  provided  should  be  evaluated,  with  special  consideration  of  the  study  design, 
reproducibility  of  data,  dose–effect  relationships  and  biological  relevance  of  the  findings.  The 
purity  of  the  test  substance  may  also  be  a  factor  to  take  into  account.  In  cases  where  an 
EU/OECD test guideline is available for a test method, the quality of a study using the method 
is regarded as being higher if it was conducted in compliance with the requirements stated in 
the test guideline. Furthermore, compared to non GLP-studies, studies compliant with GLP for 
the  same  assay  generally  provide  more  documentation  and  details  of  the  study,  which  are 
important factors to consider when assessing study reliability/quality. 

When assessing the potential mutagenicity of a substance or considering the need for further 
testing, data from various tests and genotoxic end points may be found. Both the strength and 
the weight of the evidence should be taken into account. The strongest evidence will be provided 
by  modern,  well-conducted  studies  with  internationally  established  test  protocols  and  well-
characterised test substance. For each test type and each genotoxic end point, there should be 
a separate WoE analysis. It is not unusual for positive evidence of mutagenicity to be found in 
just one test type or for only one end point. In such cases the positive and negative results for 
different end points are not necessarily conflicting but may illustrate the advantage of using test 
methods for a variety of genetic alterations to increase the probability of identifying substances 
with  mutagenic  potential.  Hence,  results  from  methods  testing  different  genotoxic  end  points 
should  not  be  combined  in  an  overall  WoE  analysis  but  should  be  subjected  to  such  analysis 
separately  for  each  end  point.  Based  on  the  whole  dataset,  one  has  to  consider  whether  an 
appropriate conclusion/assessment can be made or whether there are data gaps. If there are 
data gaps, further testing should be considered. 

3.4.2.2.  Human data 

See Section 3.1.2.2.3. 

3.4.3.  Remaining uncertainty on mutagenicity 

Reliable data can be generated  from well-designed and well-conducted studies in vitro and in 
vivo. However, due to the lack of human data available and the degree of uncertainty, which is 
inherent in testing, a certain level of uncertainty remains when extrapolating these testing data 
to the effect in humans. 

 
 
 
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3.4.4.  Concluding on suitability for risk assessment 

3.4.4.1.  Considerations on dose–response shapes and mode of action of 

mutagenic substances in test systems 

Consideration of the dose (concentration)–response effect relationship and possible mechanisms 
of action is an important component of a risk assessment. The default assumption for genotoxic 
substances  has  for  a  long  time  been  that  they  have  a  linear  dose  (concentration)–response 
relationship. However, this assumption has been challenged by experimental evidence showing 
that  both  direct  and  indirect  acting  genotoxins  can  possess  non-linear  or  threshold  dose 
(concentration)–response curves. 

Examples  of  mechanisms  of  genotoxicity  that  may  be  demonstrated  to  lead  to  non-linear  or 
threshold  dose  (concentration)–response  (effect)  relationships  include  extremes  of  pH,  ionic 
strength and osmolarity, inhibition of DNA synthesis, alterations in DNA repair, overloading of 
defence mechanisms (antioxidants or metal homeostatic controls), interaction with microtubule 
assembly leading to aneuploidy, topoisomerase inhibition, high cytotoxicity, metabolic overload 
and physiological perturbations (e.g. induction  of  erythropoiesis) (Dearfield et al., 2011).  The 
mechanisms underlying non-linear or threshold dose (concentration)–response relationships for 
some DNA reactive genotoxic substances such as alkylating agents seem to be linked to DNA 
repair capacity. 

Assessment  of  the  significance  to  be  assigned  to  genotoxic  responses  mediated  by  such 
mechanisms  would  include  assessing  whether  the  underlying  mechanism  can  be  induced  at 
substance concentrations that can be expected to occur under relevant in vivo conditions. 

In  general,  several  concentrations/doses  are  tested  in  genotoxicity  assays.  At  least  three 
experimental  concentrations/doses  have  to  be  tested,  as  recommended  in  the  OECD  test 
guidelines  for  genotoxicity.  Determination  of  experimental  dose  (concentration)-dependent 
effects is one of several pieces of experimental information that are important for assessing the 
genotoxic potential of a substance and may be used as indicated below. It should be recognised 
that not all of these considerations may be applicable to in vivo data. 

•  The  OECD  overview  document  of  the  genotoxicity  test  guidelines  (OECD,  2017)  lists  the 
relevant  criteria  to  be  fulfilled  for  a  result  to  be  considered  a  clear  positive  one:  (1)  the 
increase in genotoxic response is concentration- or dose-related, (2) at least one of the data 
points  exhibits  a  statistically  significant  increase  compared  with  the  concurrent  negative 
control and (3) the statistically significant result is outside the distribution of the historical 
negative control data (e.g. 95 %  confidence interval). In practice, the criterion  for a dose 
(concentration)-related  increase  in  genotoxicity  will  be  most  helpful  for  in  vitro  tests,  but 
care is needed to check for cytotoxicity or cell cycle delay, which may cause deviations from 
a dose (concentration)–response-related effect in some experimental systems. 

•  Genotoxicity tests are not designed to support derivation of no-effect levels. However, the 
lowest dose with an observed effect (i.e. the lowest observed effect dose) may, on certain 
occasions, be a helpful tool in risk assessment. This is true specifically for genotoxic effects 
caused  by  threshold  mechanisms,  for  example  aneugenicity.  Furthermore,  it  can  give  an 
indication of the mutagenic potency of the substance in the test at issue. Modified studies 
with additional dose or concentration points and improved statistical power may be useful in 
this  regard.  The  BMD  approach  presents  several  advantages  over  the  no  observed  effect 
dose / lowest observed effect dose approach and can be used as an alternative strategy for 
dose  (concentration)–response  assessment  (see  ECHA’s  Guidance  on  Information 
Requirements  and  Chemical  Safety  Assessment –  Chapter R.8:  Characterisation  of  dose 
[concentration]–response for human health). 

 
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•  Unusual shapes of dose (concentration)–response curves may contribute to the identification 
of specific mechanisms of genotoxicity. For example, extremely steep increases suggest an 
indirect  mode  of  action  or  a  metabolic  switching,  which  could  be  confirmed  by  further 
investigation. 

3.5.  Carcinogenicity 

The section on carcinogenicity in DWD Guidance Volume I should be considered together with 
the elements described in this section for the assessment of carcinogenicity. 

Section 1  of  Annex VI  to  Commission  Implementing  Decision  (EU)  2024/365  lists  the  criteria 
when no hazard assessment needs to be performed. 

3.5.1.  Definition 

A substance is defined as carcinogenic if it induces cancer or increases its incidence. Substances 
that  have  induced  benign  and  malignant  tumours  in  well-performed  experimental  studies  on 
animals  are  also  considered  to  be  presumed  or  suspected  human  carcinogens  unless  there  is 
strong evidence that the mechanism of tumour formation is not relevant for humans (Guidance 
on  the  Application  of  the  CLP  Criteria (60)).  Carcinogenic  substances  can  increase  the  tumour 
incidence and/or malignancy and shorten the time to tumour occurrence. Benign tumours that 
are considered to have the potential to progress to malignant tumours are generally considered 
along  with  malignant  tumours.  Substances  may  induce  cancer  by  any  route  of  exposure,  but 
carcinogenic potential and potency may depend on the conditions of exposure (e.g. route, level, 
pattern and duration of exposure). In the context of the DWD, the oral route is considered the 
most appropriate route of exposure. Carcinogens may be identified from epidemiological studies, 
animal experiments and/or other appropriate means that may include (Q)SAR analyses and/or 
extrapolation  from  structurally  similar  substances  (read-across  approach).  Each  strategy  for 
identifying  potential  carcinogens  is  discussed  in  detail  below.  The  determination  of  the 
carcinogenic  potential  of  a  substance  is  based  on  a  WoE  approach.  Classification  criteria  are 
given in the CLP regulation. 

The  process  of  carcinogenesis  involves  the  transition  of  normal  cells  into  cancer  cells  via  a 
sequence of stages that entail both genetic alterations (i.e. mutations) and non-genetic events. 
Non-genetic events are defined as those alterations/processes that are mediated by mechanisms 
that do not affect the primary sequence of DNA and yet increase the incidence of tumours or 
decrease the latency time for the appearance of tumours. For example, altered growth and death 
rates, (de)differentiation of the altered or target cells and modulation of the expression of specific 
genes associated with the expression of neoplastic potential (e.g. tumour suppressor genes or 
angiogenesis factors) are recognised to play an important role in the process of carcinogenesis 
and  can  be  modulated  by  a  chemical  agent  in  the  absence  of  genetic  change  to  increase  the 
incidence of cancer. 

Carcinogenic chemicals have conventionally been divided into two categories according to the 
presumed  mode  of  action:  genotoxic  or  non-genotoxic.  Genotoxic  modes  of  action  involve 
genetic alterations caused by the chemical interacting directly with DNA to result in a change in 
the primary sequence of DNA. A chemical can also cause genetic alterations indirectly following 
interaction  with  other  cellular  processes  (e.g.  secondary  to  the  induction  of  oxidative  stress). 
Non-genotoxic modes of action include epigenetic changes, that is, effects that do not involve 
alterations in DNA but that may influence gene expression, altered cell–cell communication, or 
affect other factors involved in the carcinogenic process. For example, chronic cytotoxicity with 
subsequent  regenerative  cell  proliferation  is  considered  a  mode  of  action  by  which  tumour 

(60)  http://echa.europa.eu/web/guest/guidance-documents/guidance-on-clp. 

 
 
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development can be enhanced: the induction of urinary bladder tumours in rats may, in certain 
cases, be due to persistent irritation/inflammation, tissue erosion and regenerative hyperplasia 
of the urothelium following the formation of bladder stones. Other modes of non-genotoxic action 
can  involve  specific  receptors  (e.g.  peroxisome  proliferator-activated  receptor  alpha  (PPARα), 
which  is  associated  with  liver  tumours  in  rodents,  or  tumours  induced  by  various  hormonal 
mechanisms). As with other non-genotoxic modes of action, these can all be presumed to have 
a threshold. 

The  objective  of  investigating  the  carcinogenicity  of  chemicals  is  to  identify  potential  human 
carcinogens, their mode(s) of action and their potency. 

Carcinogenic potential and potency information from human epidemiology studies, if available, 
requires  expert  consideration  because  of  uncertainties  in  the  exposure  assessment  and/or 
limited  sensitivity  and  statistical  power.  Reliable  human  epidemiological  studies  (e.g.  cohort 
case–control studies) can be considered using a WoE approach. In the absence of human data, 
animal  carcinogenicity  tests  may  be  used  to  differentiate  carcinogens  from  non-carcinogens. 
However, the results of these studies subsequently have to be extrapolated to humans, in both 
qualitative  and  quantitative  terms.  This  introduces  uncertainty  with  regard  to  potency  and 
relevance to humans due to species-specific factors such as differences in chemical metabolism 
and toxicokinetics and difficulties inherent in extrapolating from the high doses used in animal 
bioassays to those normally experienced by humans. 

Once a chemical has been identified as a carcinogen, there is a need to elucidate the underlying 
mode of action, that is, whether the chemical is directly genotoxic or not. In risk assessment a 
distinction is made between the different types of carcinogens - genotoxic or non-genotoxic. 

For genotoxic carcinogens exhibiting direct interaction with DNA, it is not generally possible to 
infer the position of the threshold from the no observed effect level on a dose–response curve, 
even though a biological threshold below which cancer is not induced may exist. 

For non-genotoxic carcinogens, no-effect thresholds are assumed to exist and to be discernible 
(e.g. if appropriately designed studies of the dose–response for critical non-genotoxic effects are 
conducted). No-effect thresholds may also be present for certain carcinogens that cause genetic 
alterations via indirect  effects on  DNA  following interaction with other cellular processes (e.g. 
carcinogenic risk would manifest only after chemically induced alterations of cellular processes 
had exceeded the compensatory capacity of physiological or homeostatic controls). However, in 
this  situation  the  scientific  evidence  needed  to  convincingly  underpin  this  indirect  mode  of 
genotoxic action may be more difficult to achieve. Human studies are generally not available for 
making a distinction between the abovementioned modes of action; a conclusion on this, in fact, 
depends on the outcome of mutagenicity/genotoxicity testing and other mechanistic studies. In 
addition  to  this,  animal  studies  (e.g.  the  carcinogenicity  study,  repeated  dose  studies  and 
experimental  studies  with  initiation–promotion  protocols)  may  also  inform  on  the  underlying 
mode of carcinogenic action. 

3.5.2.  Data to be used in the hazard assessment 

3.5.2.1.  Non-human data for carcinogenicity 

3.5.2.1.1. 

Non-testing data for carcinogenicity 

See Section 3.1.2.2.5 (on (Q)SARs) and Section 3.1.3 (on the read-across approach). 

 
 
 
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3.5.2.1.2. 

Testing data for carcinogenicity 

In vitro data. The following in vitro data, which provide direct or indirect information useful in 
assessing the carcinogenic potential of a substance and (potentially) the underlying mode(s) of 
action,  may  be  available.  No  single  end  point  or  effect  in  and  of  itself  possesses  unusual 
significance for assessing carcinogenic potential but must be evaluated within the context of the 
overall  toxicological  effects  of  the  substance  under  evaluation.  Standardised  protocols  do  not 
exist for most of the in vitro end points noted; rather, studies are conducted in accordance with 
expert judgement using protocols tailored to the specific substance, target tissue and cell type 
or animal species under evaluation. 

•  Genotoxicity studies. The ability of substances to induce mutations or genotoxicity can be 
indicative 
between 
mutagenicity/genotoxicity  and  carcinogenesis  are  stronger  when  effects  are  observed  in 
appropriately designed in vivo as opposed to in vitro studies. 

carcinogenic 

correlations 

potential. 

However, 

of 

•  In vitro cell transformation assay results. Such assays assess the ability of chemicals to 
induce changes in the morphological and growth properties of cultured mammalian cells that 
are  presumed  to  be  similar  to  phenotypic  changes  that  accompany  the  development  of 
neoplastic  or pre-neoplastic lesions in vivo (OECD, 2006). Similarly to in vitro assays,  cell 
transformation assays are restricted to the detection of effects of chemicals at the cellular 
level and will not be sensitive to carcinogenic activity mediated by effects exerted at the level 
of intact tissues or organisms. 

•  Mechanistic studies. Data from these studies include the following. 

o  Cell proliferation. Sustained cell proliferation can facilitate the growth of neoplastic/pre-
neoplastic cells and/or create conditions conducive to spontaneous changes that promote 
neoplastic development. 

o  Altered intercellular gap junction communication. Exchange of growth suppressive 
or other small regulatory molecules between normal and neoplastic/pre-neoplastic cells 
through  gap  junctions  is  suspected  to  suppress  phenotypic  expression  of  neoplastic 
potential. Disruption of gap junction function, as assessed by a diverse array of assays 
for  fluorescent  dye  transfer  or  the  exchange  of  small  molecules  between  cells,  may 
attenuate the suppression of neoplastic potential by normal cells. 

o  Hormone or other receptor binding. A number of agents may act through binding to 
hormone receptors or sites for regulatory substances that modulate the growth of cells 
and/or  control  the  expression  of  genes  that  facilitate  the  growth  of  neoplastic  cells. 
Interactions of this nature are diverse and generally very compound specific. 

Other targeted mechanisms of action are as follows. 

•  Immunosuppressive  activity.  Neoplastic  cells  frequently  have  antigenic  properties  that 
permit their detection and elimination by normal immune system function. Suppression of 
normal immune function can reduce the effectiveness of this immune surveillance function 
and  permit  the  growth  of  neoplastic  cells  induced  by  exogenous  factors  or  spontaneous 
changes. 

•  Ability to inhibit or induce apoptosis. Apoptosis, or programmed cell death, is a sequence 
of molecular events that results in the death of cells, most often by the release of specific 
enzymes that result in the degradation of DNA in the cell nucleus. Apoptosis is integral to the 
control of cell growth and differentiation in many tissues. Induction of apoptosis can eliminate 

 
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cells that might otherwise suppress the growth of neoplastic cells; inhibition of apoptosis can 
permit  pre-neoplastic/neoplastic  cells  to  escape  regulatory  controls  that  might  otherwise 
result in their elimination. 

•  Ability  to  stimulate  angiogenesis  or  the  secretion  of  angiogenesis  factors.  The 
growth of pre-neoplastic/neoplastic cells in solid tumours will be constrained in the absence 
of  vascularisation  to  support  the  nutritional  requirements  of  tumour  growth.  Secretion  of 
angiogenesis  factors  stimulates  the  vascularisation  of  solid  tumour  tissue  and  enables 
continued tumour growth. 

In  vitro  data  can  give  only  preliminary  information  about  the  carcinogenic  potential  of  a 
substance and possible underlying mode(s) of action. For example, in vitro genotoxicity studies 
may provide information about whether or not the substance is likely to be genotoxic in vivo, 
and thus a potential genotoxic carcinogen, and about the potential mode of action underlying 
carcinogenicity – with or without a threshold. 

Besides genotoxicity data, other in vitro data, such as in vitro cell transformation, can help to 
decide,  in  a  WoE  evaluation,  whether  a  substance  possesses  carcinogenic  potential.  Cell 
transformation results in and of themselves do not inform about the actual underlying mode(s) 
of action, since they are restricted to the detection of effects exerted at the level of the single 
cell and may be produced by mechanistically distinct processes. 

Studies  can  also  be  conducted  to  evaluate  the  ability  of  substances  to  influence  processes 
thought  to  facilitate  carcinogenesis.  Many  of  these  end  points  are  assessed  by  experimental 
systems that have yet to be formally validated and/or are the products of continually evolving 
basic research. Formalised and validated protocols are thus lacking for the conduct of these tests 
and their interpretation. Although it is difficult to give general guidance on each test due to the 
variety and evolving nature of tests available, it is important to consider them on a case-by-case 
basis and carefully consider the context in which the test was conducted. 

A  number  of  the  test  end  points  evaluate  mechanisms  that  may  contribute  to  neoplastic 
development, but the relative importance of each end point will vary as a function of the overall 
toxicological  profile  of  the  substance  being  evaluated.  It  should  also  be  noted  that  there  are 
uncertainties associated with extrapolating in vitro data to an in vivo situation, which requires 
careful consideration of the in vitro to in vivo extrapolation model used. In many cases, in vitro 
studies can offer valuable insights, which may be used in a WoE approach. 

Animal  data.  Carcinogenicity  in  experimental  animals  can  be  evaluated  using  conventional 
bioassays, bioassays that employ genetically modified animals and other in vivo bioassays that 
focus  on  one  or  more  of  the  critical  stages  of  carcinogenesis.  In  the  absence  of  data  from 
conventional long-term bioassays or assays with neoplasia as the end point, consistently positive 
results in several models that address several stages in the multistage process of carcinogenesis 
should  be  considered  in  evaluating  the  degree  of  evidence  of  carcinogenicity  in  experimental 
animals. 

Sources of information include the following. 

•  Carcinogenicity studies (conventional long-term or lifetime studies in experimental 
animals). Carcinogenicity testing is typically conducted using rats and mice, less commonly 
using animals such as the guinea pig and Syrian hamster and occasionally using minipigs, 
dogs and primates. The standard rodent carcinogenicity bioassay would be conducted using 
rats or mice randomly assigned to treatment groups. Exposures to test substances may be 
via  oral,  inhalation  or  dermal  exposure  routes.  The  selection  of  exposure  route  is  often 
dictated by a priori assumptions regarding the routes of exposure relevant to humans and/or 
other data sources (e.g. epidemiology studies or repeated dose toxicity studies in animals) 

 
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that  may  indicate  relevance  of  a  given  exposure  route.  In  the  context  of  the  DWD,  the 
appropriate  route  is  oral.  Substances that  have  induced  benign  and  malignant  tumours  in 
well-performed  experimental  studies  on  animals  are  also  considered  to  be  presumed  or 
suspected human carcinogens unless there is strong evidence that the mechanism of tumour 
formation  is  not  relevant  for  humans  (CLP  regulation,  Annex I,  Section 3.6.1.1). 
Standardised  protocols  for  such  studies  have  been  developed  and  validated  (e.g.  OECD 
TGs 451 and 453 and US EPA 870.4200). 

•  Short-  and  medium-term  bioassay  data  (e.g.  mouse  skin  tumour,  rat  liver  foci 
model,  neonatal  mouse  model).  Multiple  assays  have  been  developed  that  permit  the 
detection  and  quantitation  of  putative  pre-neoplastic  changes  in  specific  tissues.  The 
induction of such pre-neoplastic foci may be indicative of carcinogenic potential. Such studies 
are generally regarded as adjuncts to conventional cancer bioassays and, while less validated 
and  standardised,  may  be  applicable  on  a  case-by-case  basis  for  obtaining  supplemental 
mechanistic  and  dose–response  information  that  may  be  useful  for  risk  assessment 
(Enzmann et al., 1998). 

•  Genetically  engineered  (transgenic)  rodent  models  (e.g.  Xpa–/–,  p53+/–,  rasH2  or 
Tg.AC).  Animals  can  be  genetically  engineered  such  that  one  or  more  of  the  molecular 
changes  required for the multistep process  of  carcinogenesis has been accomplished.  This 
can increase the sensitivity of the animals to carcinogens and/or decrease the latency with 
which spontaneous or induced tumours are observed. The genetic changes in a given strain 
of engineered animal can increase sensitivity to carcinogenesis in a broad range of tissues or 
can  be  specific  to  the  change’s  requisite  for  neoplastic  development  in  one  or  a  limited 
number of tissues (ILSI-HESI, 2001; Pritchard  et al., 2003; Jacobson-Kram, 2004). There 
are,  thus,  limitations  in  the  interpretation  of  data  from  such  studies,  although  data  from 
these models may be used in a WoE analysis of a chemical’s carcinogenicity. 

•  Genotoxicity studies in vivo. The ability of substances to induce mutations or genotoxicity 
can be indicative of carcinogenic potential. There is, in general, a good correlation between 
positive genotoxicity findings in vivo and animal carcinogenicity bioassay results (Ashby and 
Tennant, 1991; Ashby and Paton, 1993). A decision on carcinogenicity should not be made 
from  genotoxicity  studies  alone,  but  these  studies  may  be  used  in  a  WoE  analysis  of  a 
chemical’s toxicological profile. 

•  Repeated dose toxicity tests. Such tests can identify tissues that may be specific targets 
for toxicity and subsequent carcinogenic effects. Particular  significance can be attached to 
the observation of pre-neoplastic changes (e.g. hyperplasia or metaplasia) suspected to be 
conducive  to  tumour  development,  and  the  observation  of  these  pre-neoplastic  changes 
assist in the development of dose–effect relationships (Elcombe  et al., 2002).  However, it 
should be noted that histopathological lesions in subchronic toxicity studies do not show a 
perfect correlation with the carcinogenic potential of a substance (Elcombe et al., 2002). 

•  Studies  on  the  induction  of  sustained  cell  proliferation.  Substances  can  induce 
sustained cell proliferation via compensatory processes that continuously regenerate tissues 
damaged by toxicity. Some substances can also be tissue-specific mitogens, stimulating cell 
proliferation in the absence of overt toxic effects. Mitogenic effects are often associated with 
the  action  of  tumour  promoters.  Both  regenerative  cell  proliferation  and  mitogenic  effects 
can be necessary, but not sufficient, for tumour development, but have a sufficiently different 
mechanistic basis that care should be exercised in assessing which is occurring (Cohen and 
Ellwein, 1991; Cohen et al., 1991). 

 
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•  Studies on immunosuppressive activity. As noted earlier, suppression of normal immune 
surveillance  functions  can  interfere  with  normal  immune  system  functions  that  serve  to 
identify and eliminate neoplastic cells. 

•  Studies on toxicokinetics. Such studies can identify tissues or treatment routes that may 
be  the  targets  for  toxicity  and  can  deliver  data  on  exposure  and  metabolism  in  specific 
organs. Linkages to subsequent carcinogenic impacts may or may not exist, but such data 
can serve to focus carcinogenesis studies upon specific tissue types or animal species. 

•  Other  studies  on  mechanisms/modes  of  action  (e.g.  OMICs  studies: 
toxicogenomics,  proteomics,  metabonomics  and  metabolomics).  Carcinogenesis  is 
associated  with  multiple  changes  in  gene  expression,  transcriptional  regulation,  protein 
synthesis and other metabolic changes. Specific changes diagnostic of carcinogenic potential 
have  yet  to  be  validated,  but  these  rapidly  advancing  fields  of  study  may  one  day  permit 
assessment of a broad array of molecular changes that might be useful in the identification 
of potential carcinogens. 

It is noted that the above tests inform differently on hazard identification, mode of action and 
carcinogenic potency. For example, conventional bioassays are used for hazard identification and 
potency  estimation  (i.e.  derivation  of  a  dose  descriptor),  whereas  studies  using  genetically 
engineered animals are informative on potential hazard and possibly mode of action, but less so 
on carcinogenic potency, as they are considered to be highly sensitive to tumour induction. 

In vivo data can give direct information about the carcinogenic potential of a substance, possible 
underlying mode(s) of action and its potency. 

Carcinogenicity testing should be addressed by an in vivo test in accordance with OECD TG 451 
or 453, unless the  substance is  classified as mutagen category 1A  or  1B, and a  conclusion is 
based on a comparison of the incidence, nature and time of occurrence of neoplasms in treated 
animals and controls. Other tests may contribute to a WoE evaluation, for example by providing 
supporting information or mechanistic data. 

Knowledge  of  the  historical  tumour  incidence  for  the  strain  of  animal  used  is  important 
(laboratory-specific data are preferable). Attention to the study design is also essential because 
of  the  requirement  for  statistical  analyses.  The  quality,  integrity  and  thoroughness  of  the 
reported  data  from  carcinogenicity  studies  are  essential  to  the  subsequent  analysis  and 
evaluation of studies. A qualitative assessment of the acceptability of study reports is therefore 
an important part of the process of independent evaluation. Sources of guidance in this respect 
are IEH (2002), CCCF (2004) and OECD (2002). If the available study report does not include 
all the information required by the standard test guideline, judgement is required to decide if 
the experimental procedure is acceptable or not and if essential information is lacking. 

The  final  design  of  a  carcinogenicity  bioassay  may  deviate  from  OECD  guidelines  if  expert 
judgement and experience in the testing of analogous substances supports the modification of 
protocols. Such modifications to standard protocols can be considered a function of the specific 
properties of the material under evaluation. 

Carcinogenicity data may sometimes be available in animal species other than those specified in 
standard test guidelines (e.g. guinea pig, Syrian hamster and occasionally minipigs, dogs and 
primates). Such studies may be used in addition to, or instead of, studies in rats and mice, and 
they should be considered in any evaluation. 

Data  from  non-conventional  carcinogenicity  studies,  such  as  short-  and  medium-term 
carcinogenicity assays with neonatal or genetically engineered (transgenic) animals, may also 
be available (OECD, 2002; CCCF, 2004). Genetically engineered animals possess mutations in 

 
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genes  that  are  believed  to  be  altered  in  the  multistep  process  of  carcinogenesis,  thereby 
enhancing  animal  sensitivity  to  chemically  induced  tumours.  A  variety  of  transgenic  animal 
models  exist, and new  models are continually being developed. The genetic alteration(s) in a 
specific animal model can be those suspected  of facilitating neoplastic development in a wide 
range of tissue types, or the alterations can be in genes suspected of being involved in tissue-
specific aspects of carcinogenesis. The genetic alterations must be applied while recognising both 
their experimental nature and the specific mechanistic pathways they are designed to evaluate. 
For example, a transgenic animal model sensitive to mesothelioma induction would be of limited 
value  in  the  study  of  a  suspected  liver  carcinogen.  While  such  animal  model  systems  hold 
promise for the detection of carcinogens in a shorter period of time and using fewer animals, 
their sensitivity and specificity remains to be determined. Due to a relative lack of validation, 
such  assays  have  not  yet  been  accepted  as  alternatives  to  the  conventional  lifetime 
carcinogenicity  studies,  but  existing  data  from  these  assays  may  be  useful  for  screening 
purposes or to determine the need for a rodent 2-year bioassay. Several evaluations of these 
types  of  study  have  been  published  (e.g.  ILSI-HESI,  2001;  Pritchard  et  al.,  2003;  Jacobson-
Kram, 2004). 

When  data  are  available  from  more  than  one  study  of  acceptable  quality,  consistency  of  the 
findings  should  be  established.  When  they  are  consistent,  arriving  at  a  conclusion  is  usually 
straightforward, particularly if the studies were in more than one species or if there is a clear 
treatment-related  incidence  of  malignant  tumours  in  a  single  study.  If  a  single  study  only  is 
available and the test substance is not carcinogenic, scientific judgement is needed to decide on 
whether (1) this study is relevant and (2) additional information is required to provide confidence 
that it should not be considered as carcinogenic. 

In  addition,  study  findings  may  not  clearly  demonstrate  a  carcinogenic  potential,  even  when 
approved study guidelines have been followed. For example, there may be an increase in the 
incidence of benign tumours or tumours that have a high background incidence only in control 
animals.  Although  less  convincing  than  an  increase  in  malignant  and  rare  tumours,  and 
recognising the potential oversensitivity of this model (Haseman, 1983; Ames and Gold, 1990), 
a  detailed  and  substantiated  rationale  should  be  given  before  such  positive  findings  can  be 
dismissed as not relevant. 

Repeated dose toxicity studies may provide helpful additional information to the WoE gathered 
to determine whether a substance has the potential to induce cancer and the potential underlying 
modes  of  action  (Elcombe  et  al.,  2002).  For  example,  the  induction  of  hyperplasia  (through 
cytotoxicity and regenerative cell proliferation, mitogenicity or interference with cellular control 
mechanisms)  and/or  the  induction  of  pre-neoplastic  lesions  may  contribute  to  the  WoE  for 
carcinogenic potential. However, it should be noted that histopathological lesions in subchronic 
toxicity studies do not show a perfect correlation with the carcinogenic potential of a substance 
(Elcombe  et  al.,  2002).  Toxicity  studies  may  also  provide  evidence  for  immunosuppressive 
activity, a condition favouring tumour development under conditions of chronic exposure. 

Finally, TK data may reveal the generation of metabolites with relevant structural alerts. They 
may also provide important information about the potency and relevance of carcinogenicity and 
related data collected in one species and its extrapolation to another, based upon differences in 
ADME  of  the  substance.  Species-specific  differences  mediated  by  such  factors  may  be 
demonstrated through experimental studies or by the application of TK modelling. 

Positive carcinogenic findings on animals require careful evaluation, and this should be done with 
reference to other toxicological data (e.g. in vitro and/or in vivo genotoxicity studies, TK data, 
mechanistic  studies,  (Q)SAR  evaluations)  and  the  exposure  conditions  (e.g.  route).  Such 
comparisons may provide evidence for a specific mechanism(s) of action, a significant factor to 
consider whenever possible, which may then be evaluated with respect to relevance for humans. 

 
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A  conceptual  framework  that  provides  a  structured  and  transparent  approach  to  the  WoE 
assessment of the mode of action of carcinogens has been developed (see Sonich-Mullin et al., 
2001; Boobis et al., 2006). This framework should be followed when the mechanism of action is 
key to the risk assessment being developed for a carcinogenic substance; it can be critical, in 
particular, for determining whether a substance induces cancer via genotoxic or non-genotoxic 
mechanisms. 

For  example,  a  substance  may  exhibit  limited  genotoxicity  in  vivo,  but  the  relevance  of  this 
property  to  carcinogenicity  is  uncertain  if  genotoxicity  is  not  observed  in  tissues  that  are  the 
targets  of  carcinogenesis,  or  if  genotoxicity  is  observed  via  routes  not  relevant  to  exposure 
conditions (e.g. intravenous injection) but not when the substance is administered via routes of 
administration known to induce cancer. In such instances, the apparent genotoxic properties of 
the substance may not be related to the mechanism(s) believed to underlie tumour induction. 
For example, oral administration of some inorganic metal compounds will induce renal tumours 
via a mechanism believed to involve organ-specific toxicity and forced cell proliferation. Although 
genotoxic  responses  can  be  induced  in  non-target  tissues  for  carcinogenesis  via  intravenous 
injection, there is limited evidence to suggest that this renal carcinogenesis entails a genotoxic 
mechanism (IARC, 2006). The burden of proof in drawing such mechanistic inferences can be 
high but can have a significant impact upon underlying assumptions made in risk assessment. 

In general, tumours induced by a genotoxic mechanism (known or presumed) are, in the absence 
of further information, considered to be of relevance to humans even when observed in tissues 
with no direct human equivalent. Tumours shown to be induced by a non-genotoxic mechanism 
are, in principle, also considered relevant to humans, but there is a recognition that some non-
genotoxic modes of action do not occur in humans (see OECD, 2002). This includes some specific 
types of rodent kidney, thyroid, urinary bladder, forestomach and glandular stomach tumours 
induced  by  rodent-specific  modes  of  action,  that  is,  by  mechanisms/modes  of  action  not 
operating in humans or operative in humans under extreme and unrealistic conditions. Reviews 
are available for some of these tumour types, providing a detailed characterisation that includes 
the  key  biochemical  and  histopathological  events  that  are  needed  to  establish  these  rodent-
specific  mechanisms  that  are  not  relevant  for  human  health  (IARC,  1999).  The  IPCS  has 
developed  a  framework  and  provided  some  examples  of  how  to  evaluate  the  relevance  to 
humans  of  a  postulated  mode  of  action  in  animals  (Meek  et  al.,  2003,  2014;  Boobis  et  al., 
2006;). 

The information available for substances identified as carcinogenic based on testing and/or non-
testing data should be further evaluated to identify underlying mode(s) of action and potency 
and to subsequently enable a proper quantitative risk assessment. As already pointed out, the 
use  of  existing  data  from  non-standard  animal  models  (e.g.  transgenic  or  neonatal  animals) 
needs careful evaluation, using expert judgement, of how to apply the results obtained for hazard 
and risk assessment purposes; it is not possible to provide guidance for such evaluations. 

3.5.2.2.  Human data 

See Section 3.1.4. 

3.5.3.  Remaining uncertainty 

As  indicated  in  the  previous  sections,  adequate  human  data  for  evaluating  the  carcinogenic 
properties  of  a  chemical  are  most  often  not  available,  and  alternative  approaches  have  to  be 
used. 

In  addition,  test  systems  for  identifying  genotoxic  carcinogens  are  reasonably  well  developed 
and adequately cover this property. There is also agreement that animal carcinogens that act by 
a genotoxic mode of action may be regarded as human carcinogens unless there is convincing 

 
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evidence that the mechanisms by which mutagenicity and carcinogenicity are induced in animals 
are not relevant to humans. The  relationship between carcinogenic potency in animals and in 
humans is unclear, however, and therefore introduces some uncertainty. 

There is, on the other hand, a shortage of sensitive and selective test systems to identify non-
genotoxic carcinogens, apart from the carcinogenicity bioassay. In the absence of non-testing 
information  on  the  carcinogenicity  of  structurally  related  chemicals,  indications  of  possible 
carcinogenic  properties  may  come  from  existing  repeated  dose  toxicity  data  or  in  vitro  cell 
transformation assays. However, whereas existing repeated dose toxicity data will have a low 
sensitivity (e.g. in the case of a 28-day study), for the in vitro cell transformation assay it is not 
possible  to  quantify  the  reliability  and  uncertainty  associated  with  these  assays  to  predict 
carcinogenic potential. 

Non-genotoxic carcinogens may be difficult to identify in the absence of animal carcinogenicity 
test data. However, risk assessment methodology should cover the risk for carcinogenic effects 
via this mode of action as well; current risk assessments for many non-genotoxic carcinogens 
are  based  on  NOAELs  for  precursor  effects  or  target  organ  toxicity,  with  the  application  of 
conservative AFs to address uncertainty. Once identified as a non-genotoxic carcinogen (from 
testing  or  non-testing  data)  there  may  be  uncertainty  about  the  relevance  to  humans  of  this 
observation, that is,  about the  relevance to humans of the underlying mode  of action. In the 
absence of specific data on this, observations in the animal are taken as relevant to humans. 
However, additional uncertainty will exist about the relationship between carcinogenic potency 
in  animals  and  in  humans;  this  uncertainty,  though,  will  be  addressed  in  the  procedure  for 
deriving human standards (Meek et al., 2003). 

Finally, conventional assays of carcinogenicity in animals have been found to be insensitive for 
some well-established human carcinogenic substances (e.g. asbestos and arsenic compounds). 
These substances can be shown to be carcinogenic when the test conditions are modified, thus 
illustrating that there will always be the possibility that a chemical  poses a carcinogenic hazard 
in  humans  that  was  not  detected  in  conventional  animal  studies.  This  is  also  true  for  other 
toxicological end points and should be considered in the risk assessment whenever applicable. 

3.5.4.  Concluding on suitability for risk assessment 

Besides  the  identification  of  a  chemical  as  a  carcinogenic  agent  from  either  animal  data  or 
epidemiological  data,  or  both,  dose–response  assessment  is  an  essential  further  step  in 
characterising carcinogenic risks for certain exposure conditions or scenarios. A critical element 
of this assessment is the identification of the mode of action underlying the observed tumour 
formation, whether this induction of a tumour is thought to be via a genotoxic mechanism or 
not. 

In regulatory work, it is generally assumed that, in the absence of data to the contrary, an effect 
threshold cannot be identified for genotoxic carcinogens exhibiting direct interaction with DNA, 
that is, it is not possible to define a no-effect level for carcinogenicity induced by such agents. 
However, in certain cases,  even for these  compounds, a threshold for carcinogenicity may be 
identified in the low-dose region. For example, it has in certain cases been clearly demonstrated 
that an increase in tumours did not occur at exposures below those associated with local chronic 
cytotoxicity and regenerative hyperplasia (Waddell, 2003). It is also recognised that for certain 
genotoxic  carcinogens  causing  genetic  alterations,  a  practical  threshold  may  exist  for  the 
underlying  genotoxic  effect.  For  example,  this  has  been  shown  to  be  the  case  for  aneugens 
(agents that induce aneuploidy – the gain or loss of entire chromosomes, resulting in changes 
in chromosome number) and for chemicals that cause indirect effects on DNA that are secondary 
to  another  effect  (e.g.  through  oxidative  stress  that  overwhelms  natural  antioxidant  defence 
mechanisms) (Elhajouji et al., 2011; Greim and Albertini, 2012). However, substance-specific 
evidence needs to be provided in order to demonstrate that there is a threshold for genotoxicity. 

 
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Non-genotoxic  carcinogens  exert  their  effects  through  mechanisms  that  do  not  involve  direct 
DNA reactivity. It is generally assumed that these modes of actions are associated with threshold 
doses, and it may be possible to define no-effect levels for the underlying toxic effects of concern. 
There  are  many  different  modes  of  action  thought  to  be  involved  in  non-genotoxic 
carcinogenicity. Some appear to involve direct interaction with specific receptors (e.g. oestrogen 
receptors),  whereas  others  appear  to  be  non-receptor  mediated.  Chronic  cytotoxicity  with 
subsequent  regenerative  cell  proliferation  is  considered  a  mode  of  action  by  which  tumour 
development can be induced. For example, the induction of urinary bladder tumours in rats may, 
in  certain  cases,  be  due  to  persistent  irritation/inflammation/erosion  and  regenerative 
hyperplasia of the urothelium following the formation of bladder stones, which eventually results 
in tumour formation. Specific cellular effects, such as inhibition of intercellular communication, 
have also been proposed as facilitating the clonal growth of neoplastic/pre-neoplastic cells. 

The identification of the mode of action of a carcinogen is based on a combination of results from 
genotoxicity tests (both in vitro and in vivo) and observations in animal experiments, for example 
site and type of tumour and parallel observations from pathological and microscopic analysis. 
Reliable human epidemiological studies (e.g. cohort case–control studies) can be considered in 
a WoE approach. 

Once  the  mode  of  action  of  tumour  formation  is  identified  as  having  a  threshold  or  not,  the 
following has to be considered: 

• 

• 

if the mode of action of tumour formation is defined as having a threshold, a dose descriptor 
(DNEL) has to be derived to conclude the risk assessment; 

if the mode of action of tumour formation is identified as non-threshold, a limit value (MTCtap) 
of 0.1 µg/l should be applied (see Sections 4.3 and 6.3.1). 

3.6.  Reproductive toxicity 

The section on reproductive toxicity in DWD Guidance Volume I should be considered together 
with the elements described in this section for the assessment of reproductive toxicity. 

Section 1  of  Annex VI  to  Commission  Implementing  Decision  (EU)  2024/365  lists  the  criteria 
when hazard assessment needs to be performed. 

3.6.1.  Definition 

The term reproductive toxicity is used to describe the adverse effects induced (by a substance) 
on sexual function and fertility in adult males and females, developmental toxicity in the offspring 
and effects on or mediated via lactation, as defined in part 3 of the globally harmonised system 
of  classification  and  labelling  of  chemicals.  In  practical  terms,  reproductive  toxicity  is 
characterised  by  multiple  diverse  end  points  that  relate  to  impairment  of  male  and  female 
reproductive functions or capacity (fertility) and the induction of non-heritable harmful effects 
on  the  progeny  (developmental  toxicity).  Effects  on  male  or  female  fertility  include  adverse 
effects  on  libido,  sexual  behaviour  and  any  aspect  of  spermatogenesis  or  hormonal  or 
physiological  response  that  interferes  with  the  capacity  to  fertilise,  fertilisation  itself  or  the 
development  of  the  fertilised  ovum  up  to  and  including  implantation.  Developmental  toxicity 
includes any effect interfering with normal development, both before and after birth. It includes 
effects  induced  or  manifested  either  pre-  or  postnatally.  This  includes  embryotoxic/fetotoxic 
effects  such  as  reduced  body  weight,  growth  and  developmental  retardation,  organ  toxicity, 
death, abortion, structural defects (teratogenic effects), functional effects, peri- and postnatal 
defects,  and  impaired  postnatal  mental  or  physical  development  up  to  and  including  normal 
pubertal development. 

 
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The general objectives of the testing are to establish: 

•  whether exposure of humans to the relevant chemical species can be associated with adverse 

effects on reproductive function or capacity; 

•  whether,  in  studies  in  animals,  administration  of  the  relevant  chemical  species  to  males 
and/or females prior to conception and during pregnancy and lactation causes adverse effects 
on reproductive function or capacity; 

•  whether  exposure  of  the  relevant  chemical  species  during  the  period  of  pre-  or  postnatal 

development induces non-heritable adverse effects in the progeny. 

The dose–response relationship for any substance-related adverse effects on reproduction are 
always of potential concern. It is important, however, to distinguish, where possible, between a 
primary effect on the reproductive system (due to the intrinsic property of the substance) and a 
secondary reproductive effect, usually a consequence of general toxicity (e.g. reduced food or 
water  intake,  maternal  stress).  Hence,  reproductive  toxicity  should  be  assessed  alongside 
parental  toxicity  in  the  same  study.  Further  guidance  on  the  assessment  of  developmental 
toxicity in relation to maternal toxicity is presented below. 

3.6.2.  Data to be used in the effects assessment 

3.6.2.1.  Non-human data 

3.6.2.1.1. 

Non-testing data 

For reproductive toxicity, a grouping and category approach and WoE adaptation are currently the 
best  fit-for-purpose  tools  for  non-animal  approaches  to  adapt  the  (standard)  information 
requirements for reproductive toxicity. However, appropriate justification and documentation must 
be  provided.  For  more  information,  see  Section 3.1.3  (on  the  read-across  approach)  and 
Section 3.1.4 (on WoE). 

There are a large number of potential targets/mechanisms associated with reproductive toxicity, 
which, on the basis of current knowledge, cannot normally be adequately covered by a battery 
of (Q)SAR models. For further details, see Section 3.1.2.2.5 (on (Q)SARs). 

3.6.2.1.2. 

Testing data for reproductive toxicity 

In vitro data. The design of alternatives to in vivo testing for reproductive toxicity is particularly 
challenging in view of the complexity of the reproductive process and large number of potential 
targets/mechanisms  associated  with  this  broad  area  of  toxicity.  In  addition,  many  in  vitro 
approaches do not include  elements  of biotransformation, which may differ depending  on the 
organ (Coecke et al., 2006). 

Information on the current developments in in vitro tests and methodology can be found on the 
ECVAM website ( 61) and other international centres for validation of alternative methods. ECHA’s 
website is also updated with new internationally accepted non-animal approaches (62). However, 
regulatory acceptance of these studies and approaches to replace animal testing for reproductive 
toxicity has not been achieved, as they currently do not provide equivalent information and thus 
cannot be used alone for risk assessment. In spite of this, they may support category and read 

(61)  https://joint-research-centre.ec.europa.eu/index_en. 

(62)  https://www.echa.europa.eu/support/oecd-eu-test-guidelines. 

 
 
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across  justification  and  WoE  adaptation.  They  may  also  provide  important  information  on 
mechanisms  and  modes  of  action,  or  preliminary  screening  information  that  can  be  used  in 
planning further testing. 

Currently, there are only three officially adopted EU test methods or OECD test guidelines for in 
vitro  tests  of  relevance  to  modes  of  action  for  reproductive  toxicity:  two  measuring 
oestrogenicity  (OECD  TGs 455  and  457)  and  the  other  measuring  steroidogenesis  (EU B.57 / 
OECD TG 456). Most assays under development and in the process  of  international validation 
focus on agonist/antagonistic properties measured by binding and activating or blocking a steroid 
(or a thyroid) hormone receptor. 

Three  in  vitro  embryotoxicity  tests  to  predict  developmental  toxicity  have  been  validated  but 
have  not  been  accepted  for  regulatory  use  (Genschow  et  al.,  2004;  Piersma  et  al.,  2004; 
Spielmann et al., 2004, 2006). These three tests, the  embryonic stem cell test, the limb bud 
micromass culture test and the whole embryo culture test, showed high predictivity for certain 
strongly embryotoxic chemicals. However, due to the nature of the methods and limitations in 
their predictivity, they may be used only as supporting information along with other more reliable 
data to predict developmental toxicity. The value of these validated methods could be increased 
by  incorporating  molecular-based  markers  through  the  application  of  proteomic  and 
toxicogenomic approaches (Piersma, 2006; van Dartel  et al., 2010).  The embryonic stem cell 
method may be combined with physiologically based biokinetic modelling to derive quantitative 
points of departure in vitro, which are then extrapolated to in vivo points of departure for use in 
risk assessment (Worth et al., 2014). 

The combination of assays in a tiered and/or battery approach may improve predictivity, but the 
in vivo situation remains more than the sum of the areas modelled by a series of in vitro assays 
(see Piersma (2006) for a review). Therefore, a negative result predicting absence of a particular 
property for a substance with no supporting information cannot be interpreted as demonstrating 
the  absence  of  a  reproductive  hazard  with  the  same  confidence  as  an  animal  study.  Another 
limitation of in vitro tests is that a N(L)OAEL and other dose–response information required for 
a risk assessment is not provided. 

However, a positive result predicting a particular reproductive hazard in a validated in vitro test 
could  provide  justification  for  the  need  for  further  testing  beyond  the  standard  information 
requirement,  depending  on  the  effective  concentration  and  taking  account  of  what  is  known 
about the TK profile of the substance. However, because of limited confidence in this approach 
at this time, such a result in isolation would not be adequate to support hazard classification. 

In  addition,  validated  and  non-validated  in  vitro  tests,  provided  the  applicability  domain  is 
appropriate, could be used with other data in a WoE approach to gather information on hazardous 
properties. In vitro techniques can be used in mechanistic investigations, which can also provide 
support for regulatory decisions. 

In vitro tests can also be used as supporting evidence when assessing the toxicological properties 
by  read-across  within  a  substance  grouping  approach,  providing  the  applicability  domain  is 
appropriate.  Positive  and  negative  in  vitro  test  results  may  be  of  value  in  a  read-across 
assessment and in a category approach as supporting information. 

Current  developments  in  adverse  outcome  pathways  to  build  a  combination  of  studies  and 
investigations to cover key events, from an initiating molecular event to an adverse outcome, 
may  provide  information  on  certain  pathways,  particularly  developmental  toxicity  for  certain 
malformations.  Approaches  may  combine  different  methods  (e.g.  in  vitro  tests,  (Q)SARs,  in 
chemico  assays).  As  these  pathways  do  not  cover  all  potential  mechanisms/modes  of  action, 
negative  results  predicting  absence  of  a  particular  property  from  those  approaches  do  not 
provide  enough  confidence  for  regulatory  decision-making  to  demonstrate  absence  of  a 
reproductive hazard. In addition, currently they do not provide a N(L)OAEL value or other dose–

 
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response  information  for  risk  assessment.  However,  they  may  provide  necessary  support  for 
read-across justification and categories and contribute to a WoE approach. 

Animal data. Data may be available from a wide variety of animal studies that give different 
amounts of direct or indirect information on the potential reproductive toxicity of a substance, 
for example: 

• 

screening studies (e.g. EU B.63 / OECD TG 421 or EU B.64 / OECD TG 422); 

•  one-, two- or multigeneration studies (e.g. EU B.56 / OECD TG 443, EU B.34 / OECD TG 415 

or EU B.35 / OECD TG 416); 

•  prenatal developmental toxicity tests (e.g. EU B.31 / OECD TG 414); 

•  developmental neurotoxicity studies (e.g. EU B.53 / OECD TG 426); 

• 

repeated  dose  toxicity  studies  (e.g.  EU B.7 /  OECD  TG 407  or  EU B.26 /  OECD  TG 408)  if 
relevant  parameters  are  included,  for  example  semen  analysis,  oestrous  cyclicity  and/or 
reproductive organ histopathology; 

•  male or female fertility studies of non-standard design; 

•  mechanistic and TK studies. 

Repeated dose toxicity studies 

Although not aimed directly at investigating reproductive toxicity, repeated dose toxicity studies 
(e.g. EU B.7 / OECD TG 407 or EU B.26 / OECD TG 408) may reveal clear effects on reproductive 
organs in adult animals. However, if these findings occur in the presence of marked systemic 
toxicity (up to the highest dose level tested in a repeated dose study), it may lower concerns for 
effects on fertility and can contribute to decisions on further testing requirements. However, this 
does not rule out the possibility that the substance may have the capacity to affect fertility. 

The observation of effects on reproductive organs in repeated dose toxicity studies may also be 
sufficient for identifying a N(L)OAEL for use in the risk assessment. It should, however, be noted 
that the sensitivity of repeated dose toxicity studies for detecting effects on reproductive organs 
may  be  less  than  that  of  reproductive  toxicity  studies  (e.g.  extended  one-generation 
reproductive toxicity studies) because of the lower number of animals per group. Some effects 
seen in repeated dose toxicity studies may be difficult to interpret, for example changes in sex 
hormone level, and should be investigated further as part of other studies. If there are indications 
of adverse effects on the reproductive organs of adult animals in repeated dose toxicity studies, 
further  studies  may  be  triggered  (for  details  on  the  triggers,  consult  Section 6.9  of  DWD 
Guidance  Volume I).  Repeated  dose  toxicity  studies  may  also  provide  indications  on  potential 
developmental  neurotoxicity  and/or  developmental  immunotoxicity,  which  may  need  to  be 
further investigated. 

In vivo reproductive toxicity tests 

The available OECD test guidelines specifically designed to investigate reproductive toxicity are 
shown in Table 10. 

The purpose of the reproduction/developmental toxicity screening test (EU B.63 / OECD TG 421 
and  EU B.64 /  OECD  TG 422)  is  to  provide  information  on  the  effects  on  male  and  female 
reproductive performance in rodents, such as gonadal function, mating behaviour, conception, 
development of conceptus and parturition. The observation in these tests of clear evidence of 
adverse effects on reproduction or reproductive organs may be sufficient to meet the information 

 
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needs for a classification and risk assessment (using an appropriate AF) and provide a N(L)OAEL 
from which a DNEL can be identified. The results should be interpreted with caution, because 
OECD TGs 421 and 422 are screening assays that were not designed as an alternative to or a 
replacement for the definitive reproductive toxicity studies (EU B.34 / OECD TG 415, EU B.56 / 
OECD  TG 443  and  EU B.35 /  OECD  TG 416).  These  screening  tests  are  not  meant  to  provide 
complete  information  on  all  aspects  of  reproduction  and  development.  In  particular,  the 
postnatal  effects  associated  with  prenatal  exposure  (e.g.  undetected  malformations  affecting 
viability or functional effects) or effects resulting from postnatal or lactational exposure are not 
covered  in  these  studies.  Furthermore,  the  exposure  duration  in  these  studies  may  not  be 
sufficient to detect all effects on the spermatogenic cycle, although it is likely that in practice the 
2-week exposure period will be sufficient to detect the majority of testicular toxicants (Ulbrich 
and Palmer, 1995). However, the number of animals per dose group is limited, which may affect 
the statistical power of the study to detect an effect. These screening tests may in some cases 
give  indications  of  reproductive  effects  (e.g.  fertility  and  postnatal  effects)  that  cannot  be 
investigated in a prenatal developmental toxicity study (EU B.31 /  OECD TG 414). A negative 
result in a screening study may lower concerns for reproductive toxicity, but this will not provide 
reassurance  about  the  absence  of  this  hazardous  property.  However,  a  negative  result  can 
provide the basis for DNEL derivation in relation to reproductive toxicity derived from the highest 
dose level used in the study and using an AF that takes account of the limitations of this study. 
An evaluation of EU B.63 / OECD TG 421 and EU B.64 / OECD TG 422 has confirmed that these 
tests  are  useful  for  initial  hazard  assessment  and  can  contribute  to  decisions  on  further  test 
requirements (Reuter et al., 2003; Gelbke et al., 2004). 

The two-generation study (EU B.35 / OECD TG 416) is a general test that enables evaluation of 
the effects of the test substance on the complete reproductive cycle, including libido, fertility, 
development  of  the  conceptus,  parturition,  postnatal  effects  in  both  dams  (lactation)  and 
offspring, and the reproductive capacity of the offspring. 

The extended  one-generation reproductive toxicity study (EU B.56 / OECD TG 443) addresses 
the main limitation of EU B.34 / OECD TG 415 by incorporating additional postnatal evaluations, 
which  include  clinical  pathology,  a  functional  observation  battery,  immunotoxicity  end  points, 
oestrous cyclicity and semen analysis, and, using an extended F1 generation dosing period (to 
postnatal  day 70),  end  points  addressing  developmental  neurotoxicity.  The  study  has  a 
shortened F0 male premating dosing period, justified by the observation of no differences in the 
detection rates for adverse effects on fertility between 4- and 9-week premating dosing periods 
in a number of studies (reviewed by Ulbrich and Palmer, 1995). 

The  prenatal  developmental  toxicity  study  (EU B.31 /  OECD  TG 414)  provides  a  focused 
evaluation  of  potential  effects  on  prenatal  development,  although  only  effects  that  are 
manifested before birth can be detected. 

Positive results in these studies will be relevant to hazard classification and the human health 
risk assessment, unless there is information to show that effects seen in these studies are not 
relevant for humans. N(L)OAELs can be identified from all studies discussed above. 

Developmental neurotoxicity studies (EU B.53 / OECD TG 426 and EU B.56 / OECD TG 443) are 
designed  to  provide  information  on  the  potential  functional  and  morphological  hazards  to  the 
nervous  system  arising  in  the  offspring  from  exposure  of  the  mother  during  pregnancy  and 
lactation.  These  studies  investigate  changes  in  behaviour  due  to  effects  on  the  CNS  and  the 
peripheral nervous system. As behaviour may also be affected by the function of other organs 
such as the liver, kidneys and the endocrine system, toxic effects on these organs in offspring 
may also be reflected in general changes in behaviour. No single test is able to reflect the entire 
complex  and  intricate  function  of  behaviour.  For  testing  behaviour,  therefore,  a  range  of 
parameters in a functional test battery, is used to identify changes in individual functions. 

 
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The severity and nature of the effect should be considered. Generally, a pattern of effects (e.g. 
impaired  learning  during  several  consecutive  trials)  is  more  persuasive  evidence  of 
developmental  neurotoxicity  than  one  or  a  few  unrelated  changes.  The  reversibility  of  effects 
should be considered, too. Irreversible effects are clearly serious, while reversible effects may 
be  of  less  concern.  However,  it  is  often  not  possible  to  determine  whether  an  effect  is  truly 
reversible. The nervous system possesses reserve capacity, which may compensate for damage, 
but  the  resulting  reduction  in  reserve  capacity  should  be  regarded  as  an  adverse  effect.  If 
developmental neurotoxicity is observed only during some of the lifespan, then compensation 
should  be  suspected.  In  addition,  effects  observed,  for  example,  during  the  beginning  of  a 
learning  task  but  not  at  the  end  should  not  be  interpreted  as  reversible  effects.  Rather,  the 
results may indicate that the speed of learning is decreased. 

The  experience  of  offspring,  particularly  during  infancy,  may  affect  their  later  behaviour.  For 
example, frequent handling of rats during infancy may alter the physiological response to stress 
and the behaviour in tests for emotionality and learning. In order to control for environmental 
experiences, the conditions under which the offspring are reared should be standardised within 
experiments  with  respect  to  variables  such  as  noise  level,  handling  and  cage  cleaning.  The 
performance of the animals during the behavioural testing may be influenced by, for example, 
the time of day and the stress level of the animals. Therefore, the most reliable data are obtained 
in  studies  where  control  and  treated  animals  are  tested  alternatively  and  environmental 
conditions are standardised. 

Equivocal  results  may  need  to  be  followed  up  by  further  investigation.  The  most  appropriate 
method  for  further  investigation  should  be  determined  on  a  case-by-case  basis.  Additional 
guidance  is  provided  in  DWD  Guidance  Volume I  and  ECHA’s  Guidance  on  Information 
Requirements  and  Chemical  Safety  Assessment,  Chapter R.8,  and  Guidance  on  the  Biocidal 
Products Regulation – Volume III: Human health – Part A: Information requirements ( 63). 

Positive results in a developmental neurotoxicity study will be relevant to hazard classification 
and  the  human  health  risk  assessment,  providing  a  N(L)OAEL,  unless  there  is  information  to 
show that effects seen in these studies are not relevant for humans. 

For  more  detailed  reviews  of  how  to  interpret  the  test  guidelines  mentioned  in  this  section, 
including discussions of their strengths and limitations, see the reports from ECETOC (2002) and 
WHO (2001). 

Table 10. Overview of in vivo OECD test guidelines for reproductive toxicity 

Test 

Design 

End points 

EU B.56 / OECD TG 443: 
extended one-
generation study 

Exposure of 10 weeks (unless specific 
reasons to shorten) prior to mating (P) and 
until postnatal day 90–120 (cohorts 1A and 
1B). If the extension of cohort 1B is 
triggered, then until postnatal day 4 or 21 
(F2) 

Three dose levels plus control; highest dose 
level must be chosen with the aim of 
inducing some systemic toxicity but not 
death or severe suffering 

Preferred species: rat 
Preferred route: oral 
N = sufficient mating pairs to produce 20 

Parental (P) generation: 

Growth, survival and fertility 

Oestrus cyclicity and sperm 
quality 

Pregnancy length and litter 
size 

Histopathology and weight 
of reproductive and non-
reproductive organs 
Haematology and clinical 
chemistry 

Offspring (F1): 

(63)  ttps://echa.europa.eu/documents/10162/2324906/bpr_guidance_vol_iii_part_a_en.pdf/05e4944d-106e-9305-

21ba-f9a3a9845f93?t=1648525287369. 

 
 
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Test 

Design 

pregnant animals per dose group (P 
generation) 
N = 20 mating pairs (extension of 
cohort 1B, if triggered) 

N = 10 males and 10 females per dose 
group (cohorts 2A, 2B and 3, if triggered) 

EU B.35 / OECD TG 416: 
two-generation study 

Exposure before mating for at least 
10 weeks until the end of the mating period 
(males) until weaning of second generation 
(females) 

At least three dose levels (unless limit test) 
plus control 

Sufficient number of animals to yield 
preferably not less than 20 pregnant 
females per dose group 

Preferred species: rat 

EU B.34 / OECD TG 415: 
one-generation study 

Males: exposure before mating for at least 
one spermatogenic cycle until end of mating 
period 

Females: exposure before mating for at 
least 2 weeks until weaning of first 
generation 

At least three dose levels (unless limit test) 
plus control 

Sufficient number of animals to yield about 
20 pregnant females per dose group 

Species: rat or mouse 

End points 

Growth, survival and sexual 
maturation 
Histopathology and weight 
of reproductive and non-
reproductive organs 
(cohort 1A) 
Weight of reproductive 
organs and optional 
histopathology (cohort 1B) 
Haematology and clinical 
chemistry 

Fertility of F1 animals to 
produce F2 generation 
(extension of cohort 1B) 
under certain conditions 
Developmental neurotoxicity 
(cohorts 2A and 2B or a 
separate study) in case of a 
particular concern 
Developmental 
immunotoxicity (cohort 3 or 
a separate study) in case of 
a particular concern 

Fertility 

Oestrus cyclicity and sperm 
quality 
Pregnancy outcome (e.g. 
dystocia) 

Growth, development and 
viability 

Anogenital distance if 
triggered 

Sexual maturation 
Histopathology and weight 
of reproductive organs, 
brain and target organs 
Recommended: motor 
activity, sensory function 
and reflex ontology in F1 
generation 

Fertility 

Growth, development and 
viability 
Histopathology and weight 
of reproductive organs, 
brain and target organs 

EU B.31 / OECD TG 414: 
prenatal developmental 

Exposure at least from implantation to 1–
2 days before expected birth 

Clinical observations 

Body weight and food/water 

 
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Test 

Design 

toxicity study 
(teratology study) 

EU B.53 / OECD TG 426: 
developmental 
neurotoxicity study 

At least three dose levels (unless limit test) 
plus control 
Sufficient number of females to result in 
approximately 20 female animals with 
implantation sites 

Preferred rodent species: rat 

Preferred non-rodent species: rabbit 

Exposure at least from implantation and 
throughout lactation (postnatal day 20) 

Three dose levels plus control 

N = 20 pregnant females 

EU B.63 / OECD TG 421 
and EU B.64 / OECD 
TG 422: reproduction/ 
developmental toxicity 
screening test  

Males: exposure before mating for at least 
2 weeks until end of mating period 
Females: exposure before mating for at 
least 2 weeks until at least postnatal 
day 13. 
At least three dose levels (unless limit test) 
plus control 

At least 10 males and 12–13 females per 
group 

91 

End points 

consumption 

Implantation and 
resorptions 

Fetal growth 

Morphological variations and 
malformations 

Birth and pregnancy length 

Growth, development and 
viability 
Physical and functional 
maturation 
Behavioural changes due to 
central and peripheral 
nervous system effects 
Brain weights and 
neuropathology 

Clinical observations 

Body weight and food/water 
consumption 

Fertility 

Pregnancy length and birth 

Thyroid hormones (T4) 

Clinical chemistry 

Gross necropsy 

Organ weights 

Histopathology 
Fetal and pup growth and 
survival until day 4 

NB: 

T4, thyroid hormone thyroxine. 

Developmental  effects  should  be  considered  in  relation  to  adverse  effects  occurring  in  the 
parents. Since adverse effects in pregnancy or the postnatal period may result as a secondary 
consequence of maternal toxicity, reduced food or water intake, maternal stress, lack of maternal 
care,  specific  dietary  deficiencies,  poor  animal  husbandry,  intercurrent  infections,  etc.,  it  is 
important  that  the  effects  observed  should  be  interpreted  in  conjunction  with  possible 
concomitant maternal toxicity (ECB, 2004; Fleeman  et  al.,  2005;  Cappon  et  al.,  2005). The 
nature,  severity  and  dose–response  of  all  effects  observed  in  progeny  and  parental  animals 
should be considered and compared to achieve a balanced integrated assessment of available 
data on all end points relevant for reproductive toxicity. 

3.6.2.1.3. 

Relation between maternal toxicity and developmental toxicity 

Developmental  toxicity  may  be  mediated  by  maternal  toxicity,  as  experience  has  shown. 
However, in most studies, evidence for a causal relationship is lacking. Developmental toxicity 
occurring in the presence of maternal effects does not itself imply a causal relationship between 
the two; therefore, it is not appropriate to discount developmental toxicity that occurs only in 
the  presence  of  maternal  toxicity.  Developmental  effects  that  occur  even  in  the  presence  of 
maternal  toxicity  are  considered  to  be  evidence  of  developmental  toxicity,  unless  it  can  be 
unequivocally  demonstrated  on  a  case-by-case  basis  that  the  developmental  effects  are 
secondary to maternal toxicity. 

 
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For  the  assessment  of  the  interrelationship  between  developmental  toxicity  and  maternal 
toxicity, please refer to Section 3.7.2.4 of Annex I to Regulation (EC) No 1272/2008 (the CLP 
regulation) ( 64) and Guidance on the Application of the CLP Criteria ( 65). 

3.6.2.1.4. 

Reproductive toxicity via lactation 

Reproductive toxicity may occur through lactation in several ways. Substances may reach the 
milk and result in exposure of the newborn. Alternatively, the quality and quantity of the milk 
may be affected by maternal exposure to the substance, resulting in nutritional effects on the 
newborn.  Three  aspects  are  crucial  in  the  risk  assessment  of  lactational  effects,  as  indicated 
below. 

•  The concentration of the substance transferred via the milk. TK aspects should be considered, 
including  the  chemico-physical  properties  of  the  compound,  the  timing  and  duration  of 
exposure, the bioavailability and the persistence of the substance. Fat-soluble chemicals that 
may be mobilised during lactation are of particular concern. 

•  The sensitivity of the newborn compared with the adult. A wide spectrum of toxic effects may 
occur  in  the  newborn,  ranging  from  general  toxic  effects,  which  may  present  as  reduced 
weight gain or delayed general development, to specific effects on the maturation of organs 
or  physiological  systems.  The  newborn  may  be  more  sensitive  than  the  adult,  not  only 
because of specific developmental end points, but also in view of a possibly higher intake of 
the  substance  per  kilogram  of  body  weight  and  the  immaturity  of  detoxification  pathways 
and physiological barriers. Moreover, some effects may become apparent only later in life. 

•  Effects on milk quality and/or quantity. These effects will usually be detected only through 
effects on the growth and development of the newborn. Any effect on the quantity or quality 
of  the  breast  milk  is  likely  to  be  due  to  systemic  effects  in  the  mother.  However,  overt 
maternal  toxicity  may  not  be  seen  (e.g.  the  substance  may  affect  only  the  transfer  of  a 
nutrient into the milk, with no consequence for the mother). If a substance causes marked 
overt systemic toxicity in the mother at the same dose level, then it is possible that this may 
indirectly impair milk production or impair maternal care as a non-specific secondary effect. 
The type and magnitude of the maternal effects and their potential influence on lactation / 
milk production needs to be considered on a case-by-case basis using expert judgement. In 
general, the two-generation study (EU Annex V B.35 or OECD TG 416) or the extended one-
generation  study  (OECD  TG 443)  are  the  best  guideline-based  studies  available  for 
identifying effects on or via lactation. In the case of specific questions regarding lactation, 
the protocol may have to be amended in view of any existing information on the substance 
under  study,  including  physico-chemical,  TK  and  general  toxic  properties.  Cross-
fostering (66) may establish whether toxicity to the offspring is the result of lactational effects 
or uterine exposure. 

(64)  https://eur-lex.europa.eu/eli/reg/2008/1272/oj. 

(65)  https://echa.europa.eu/documents/10162/2324906/clp_overview_en.pdf/515227da-8a2e-c554-1320-

ee20e7cccb7c?t=1730719032697.  

(66)  Cross-fostering is a technique used in animal husbandry, animal science, genetic and nature versus nurture studies, 
and conservation, whereby offspring between two predefined groups are usually exchanged after birth, thus being 
removed from their biological parents and raised by surrogates. 

 
 
 
 
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3.6.2.2.  Human data 

See Section 3.1.2.2.3. 

93 

3.6.3.  Remaining uncertainty on reproductive toxicity 

Unless the effect is a very specific one of low ‘normal’ incidence, there may be a high level of 
uncertainty in human studies of effects on reproduction (see Section 3.1.2.2.3). 

It is obvious that there are limitations in many of the types of non-human studies relating to 
reproductive  toxicity.  Well-conducted  tests  following  EU B.35/B.31  or  OECD  TG 416/414 
standards can be used with confidence to identify whether substances are toxic to reproduction 
or not in relation to the end points addressed in the test. However, other studies, including tests 
conducted in accordance with EU B.63/B.64 or OECD TG 421/422, may provide clear (in the case 
of  the  OECD  methods)  or  indicative  evidence  of  reproductive  toxicity,  but  will  not  provide 
sufficient evidence for confidence about the absence of reproductive toxicity. The WoE from other 
studies (including human, TK and/or mechanistic data), when available, can help in reducing this 
uncertainty. 

3.6.4.  Conclusions on reproductive toxicity 

Reproductive  toxicity  end  points  should  be  considered  collectively,  using  a  WoE  approach  to 
establish  the  most  relevant  end  point  and  its  NOAEL  or  critical  effect  dose  to  be  used  in  risk 
assessment. 

A WoE assessment involves the consideration of all data that are available and may be relevant 
to reproductive toxicity. There can be no firm rules for conducting a WoE assessment, as this 
process involves expert judgement and because the mix and reliability of information available 
for  a  particular  substance  will  probably  be  unique.  In  addition,  the  WoE  assessment  should 
consider all toxicity end points together, and not look at reproductive toxicity in isolation. Further 
recommendations on how to perform WoE are given in the EFSA guidance on the use of the WoE 
approach ( 67), and Chapter R.4 of the ECHA guidance (68) can be followed. 

One example of a WoE assessment is the pooling of information from several available in vivo 
reproductive  toxicity  studies.  Individually,  these  studies  may  have  deficiencies,  such  as  brief 
reporting, small group size, limited range of end points evaluated, the dose levels or the dosing 
schedule  not  being  appropriate  for  a  comprehensive  evaluation  of  potential  effects  on  the 
reproductive  cycle,  and  the  study  not  complying  with  GLP.  However,  taking  account  of  their 
reliability and relevance and consistency of findings, collectively these studies could provide a 
level of information similar to that of the EU or OECD test guideline studies, and therefore meet 
the information requirements needed for the classification decision and risk assessment. 

3.6.5.  Concluding on suitability for risk assessment 

In  order  to  be  suitable  for  risk  assessment,  appropriate  threshold  levels  (NO(A)EL/LO(A)EL, 
BMD) have to be established. Reproductive effects due to endocrine disrupting mode of action 
are regarded as non-threshold effects, and a limit value (MTCtap) of 0.1 µg/l should be applied 
instead (see Sections 4.3 and 6.3.1). 

(67)  https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2017.4971. 

(68)  https://echa.europa.eu/documents/10162/17235/information_requirements_r4_en.pdf/d6395ad2-1596-4708-

ba86-0136686d205e?t=1323782558175. 

 
 
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4.  Hazard assessment: hazard characterisation (dose–
response / concentration-response relationship) 

4.1.  Introduction 

For the derivation of a  DNEL, all available hazard information  regarding systemic toxicity and 
local  effects  at  the  port  of  entry  (e.g.  GI  tract)  needs  to  be  evaluated  (see  Chapter 3  of  this 
guidance document) and, where possible, dose descriptors (BMD, N(L)OAEL,  etc.) need to be 
established. The following is noted: 

•  a systemic effect is defined as an effect that is normally observed distant from the site of 
first contact, that is, after having passed through a physiological barrier (mucous membrane 
of the GI tract or of the respiratory tract, or the skin), and becomes systemically available; 

•  a  local  effect  is  an  effect  that  is  observed  at  the  site  of  first  contact  (GI  mucosa –  in  the 
context of the DWD), caused irrespective of whether a substance is systemically available. 

It should be noted, however, that toxic effects on surface epithelia may reflect indirect effects 
as a consequence of systemic toxicity or secondary to systemic distribution of the substance or 
its active metabolite(s). Figure 4 provides a schematic representation of the steps for performing 
hazard characterisation. 

Hazard characterisation involves the following steps. 

• 

Identification of critical effects (outcome of the hazard identification). 

•  For effects where a threshold is identified: 

o  dose–response  assessment –identification  of  most  relevant  dose  descriptor  (e.g. 

N(L)OAEL/BMD); 

o  modification  of  relevant  dose  descriptor –  application  of  appropriate  AFs  to  the  dose 

descriptor to address uncertainties (see Section 4.2.2); 

o  derivation of systemic DNEL. 

•  For effects where a threshold is not identified (see also Chapter 6): 

o 

o 

if the relevant chemical species falls into the lowest migration tier (only genotoxicity data 
provided) and it is non-genotoxic, then MTCtap = 2.5 µg/l; 

if  the  assessment  identifies  carcinogenicity  and/or  mutagenicity  and/or  ED  hazard, 
irrespective of the migration tier, and there is no robust scientific evidence of an existing 
threshold, then an MTCtap of 0.1 µg/l must apply (see Section 6.2.3). 

 
 
 
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95 

Figure 4. Schematic representation of the steps for performing the hazard assessment 

Identification of critical effects

• 
• 
• 

Consider both systemic and route-specific (oral) local effects (GI irritation)
Establish most relevant animal species and study
Consider dose–response relationships

Yes

Is the relevant 
chemical species 
genotoxic?

No

Is information 
available on 
genotoxicity only 
(due to low 
migration tier)?

No

Does the relevant 
chemical species 
show any other 
non-threshold 
effect (Carc or ED)?

No

Dose response assessment

Yes or data insufficient

Yes

Relevant chemical 
species is not 
genotoxic at low 
migration band

See Section 6.3.1 of 
DWD Guidance 
Volume II

(MTCtap = 2.5 µg/l)

Identification of dose 
descriptor for 
systemic effects

Identification of dose 
descriptor for local 
effects (GI irritation)

Carc or Muta or ED 
hazard concern 
identified

Select effect with 
relevant LOAEL; use 
respective NOAEL as 
starting point for 
DNEL

Local effects at the 
site of entry (GI 
tract).
Give as relevant 
external concentration

See Sections 6.2.2 
and 6.2.3 of DWD 
Guidance Volume II

(MTCtap = 0.1 µg/l)

Modification of dose descriptor

Application of appropriate AF

Derivation of Systemic DNELs

Establish MTCtap value

(Sections 6.3 and 6.4 of DWD Guidance Volume II)

 
 
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4.2.  Identification of critical effects 

In  the  first  step  of  hazard  assessment,  the  whole  data  package  should  be  evaluated  for 
assessment  of  the  most  relevant  critical  effects,  considering  the  biological  plausibility  of  the 
dose–effect  relationship,  its  consistency  over  the  whole  data  package,  the  severity  and 
reversibility of the effect, and the mode of action (if known) and its relevance for humans. For 
the latter, the IPCS developed a framework for analysing the relevance of a non-cancer (Boobis 
et al., 2008) or cancer mode of action for humans (Boobis et al., 2006). The IPCS framework on 
mode  of  action /  species  concordance  analysis  was  updated  to  take  into  account  new 
developments  in  the  field  of  risk  assessment  (Meek  et  al.,  2003).  This  framework  gives  the 
opportunity to present in a transparent manner the evidence for the key events leading to an 
adverse effect and to identify a causal linkage (through dose–response and time concordance). 

Furthermore, the data package should be evaluated with respect to local effects at the port of 
entry.  If,  instead  of  an  oral  study,  an  inhalation  study  is  used  to  cover  the  repeated  dose 
information  requirement  for  substances  under  the  DWD,  local  effects,  such  as  lesions  in  the 
airways in inhalation studies or on the skin in dermal studies, should not be considered relevant 
for  derivation  of  a  threshold  for  local  toxicity.  Only  local  effects  in  the  GI  tract  need  to  be 
considered. 

Before deriving a DNEL on the basis of the dose descriptors, it is important to determine whether 
the  relevant  chemical  species  exerts  its  effects  by  a  non-threshold  mode  of  action  (e.g.  non-
threshold  mutagens,  carcinogens,  EDs)  or  whether  a  threshold  is  possible  to  derive  (e.g. 
systemic toxicity, reproductive toxicity). 

If the substance exerts its effects by a threshold mode of action, a DNEL value must be derived 
for the most critical effect(s) (see Section 4.2.2). 

When the decision on a threshold and a non-threshold mode of action cannot be unambiguously 
made, the non-threshold mode of action should be assumed. 

4.2.1.  Hazard information underlying the derivation of a derived no-effect 

level 

4.2.1.1. 

Toxicokinetics and absorption 

Data on TK will provide information on the possible fate of the substance in the human body. 
Sufficient information on absorption should be available to support route-to-route extrapolation 
in  the  risk  characterisation  where  it  is  needed  or  to  address  species-specific  mechanisms  if 
relevant. Furthermore, absorption differences between species may be taken into consideration 
for modification of the dose descriptor during the risk assessment (see Section 4.2.2.2). 

4.2.1.2. 

Systemic (target organ) toxicity after repeated dose 
administration 

Information  on  systemic  (target  organ)  toxicity  after  repeated  dose  administration  can  be 
derived from short-term (28-day), subchronic (90-day) and chronic/carcinogenicity studies. 

Most  systemic  (target  organ)  effects  can  be  assessed  using  quantitative  risk  characterisation 
and therefore depend upon the difference in dose levels at which adverse  effects are  seen in 
animals  (or  humans)  and  the  estimated  exposure  for  the  substance.  The  key  factors  are  the 
most sensitive, relevant NOAEL, the effects it is based upon and the dose response that occurs 
at higher doses. 

 
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4.2.1.3. 

Toxicity to reproduction and development 

Effects  on  the  reproductive  system  are  often  threshold  based,  allowing  a  quantitative  risk 
characterisation to be carried out. There could be, however, some exceptions, in particular when 
non-threshold  endocrine  disrupting  mode  of  action  or  genotoxic  mechanisms  underly  the 
reproductive effects or the effects on the development of offspring. In such cases, an MTCtap of 
0.1 µg/l should be applied. 

If  the  DNEL  is  based  on  severe  reproductive  effects,  the  need  for  an  additional  AF  should  be 
considered. The AF will depend upon the severity of effects, their relationship to toxicity observed 
in  the  mothers  and  the  exposure  level  at  which  they  occurred  compared  with  effects  seen  in 
other animals. 

Fertility  and  developmental  effects  are  relevant  end  points  for  exposure  scenarios  involving 
repeated exposure to contaminants in drinking water. 

4.2.1.4. 

Other toxicity end points 

In  addition  to  the  abovementioned  effects,  other  effects,  such  as  neurotoxicity  and 
immunotoxicity, must be considered. They may be reversible or irreversible. In either case, the 
acceptability of the effects will be reflected by the relevant DNEL accounting for oral, long-term 
exposure. 

4.2.2.  Derived no-effect level derivation for threshold effects 

A dose below which no adverse effect will occur exists for most types of toxicity, according to 
current knowledge. For relevant chemical species that have such dose limit, a DNEL value should 
be established. The most sensitive end point in the most relevant oral study should be used in 
the derivation of a DNEL, as follows: 

where: 

𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 =

𝐷𝐷𝑁𝑁𝑁𝑁𝐷𝐷𝐷𝐷 𝑜𝑜𝑜𝑜 𝐷𝐷𝑁𝑁𝑁𝑁𝐷𝐷𝐷𝐷 𝑜𝑜𝑜𝑜 𝐵𝐵𝐵𝐵𝐷𝐷𝐷𝐷
𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝐴𝐴

•  NOAEL = no observed adverse effect level; 

•  LOAEL = lowest observed adverse effect level; 

•  BMDL = lower confidence limit on the benchmark dose; 

•  AF = assessment factor. 

The  DNEL  value  is  used  further  to  calculate  the  maximum  concentration  at  tap  (MTCtap)  (see 
Section 4.1). Usually, the study in the most sensitive and relevant animal species resulting in 
the most relevant lowest dose descriptor (e.g. BMDs, NOAELs, NOAECs, LOAELs, LOAECs) will 
be  selected  for  DNEL  derivation.  Often,  several  studies  addressing  a  certain  end  point  are 
available  for  one  relevant  chemical  species.  Different  dose  spacing  in  these  studies  results  in 
different  dose  descriptors  (e.g.  BMDs,  NOAELs,  LOAELs).  If  study  design  and  end  points 
addressed are comparable, it might be appropriate to consider these studies together. Special 
attention should be paid to the relevance of effects, animal species and exposure duration when 
choosing the dose descriptor, and the choice must be justified. 

 
 
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As a general rule, if several relevant NOAELs (or other dose descriptors) are available, the one 
that would result in the lowest DNEL should be chosen. However, the lowest dose descriptor may 
not  always  provide  the  lowest  DNEL  value,  as  it  depends  on  the  AFs  that  will  be  used  for  its 
derivation. Therefore, the choice of the critical dose descriptor should be made bearing in mind 
that  the  resulting  DNEL  should  also  be  the  critical  one  for  use  in  risk  characterisation.  The 
duration of the study should also be considered when deciding on the critical dose descriptor. In 
general, the experimental NOAEL will decrease with increasing exposure times and other, and 
more serious, adverse effects may appear with increasing exposure times. Consequently, to end 
up with the most conservative DNEL for repeated dose toxicity, chronic exposure is the ‘worst 
case’. Therefore, if an appropriate chronic toxicity study is available, this is the preferred starting 
point, and no AF for duration extrapolation is needed. If only a subacute or subchronic toxicity 
study is available, appropriate AFs must be applied (see Section 4.2.2.3). 

Alternatively,  the  assessor  should  derive  all  possible  DNELs  from  the  available  relevant  dose 
descriptors  for  each  end  point  and  then  choose  the  most  critical  DNEL  to  apply  in  the  risk 
assessment. 

4.2.2.1. 

Relevant time frames in DNEL derivation 

A DNEL for the general population’s long-term exposure to substances used in materials coming 
into contact with drinking water can be derived. 

Toxicity  studies  that  give  information  on  these  possible  long-term  effects  of  a  substance  are 
repeated  dose  toxicity  studies,  reproductive  toxicity  studies  (including  developmental  toxicity 
studies) and carcinogenicity studies. Here, ‘long-term’ is used as a more general term, including 
subchronic (usually 90 days) as well as chronic (usually 1.5–2 years) studies. 

When  valid  developmental  toxicity  studies  are  available,  all  relevant  critical  effects  should  be 
evaluated  together  with  other  observations  from  other  studies.  If  the  dose  descriptor  (e.g. 
NOAEL) derived from relevant effects in a valid developmental toxicity study is lower than that 
from a short-term repeated dose toxicity study and this cannot be explained by dose spacing, 
the dose descriptor (e.g. NOAEL) from the developmental toxicity study should be used for the 
derivation  of  the  DNEL  value.  This  will  apply  to  the  general  population  (thus  protecting  both 
pregnant and non-pregnant women). Developmental studies are often the only studies that use 
gavage dosing with the aim of determining a dose descriptor (e.g. NOAEL). Maternal effects can 
be  regarded  as  critical  effects  for  deriving  a  long-term  DNEL  if  they  are  deemed  relevant  in 
comparison with other critical effects observed in other valid repeated dose toxicity studies. 

4.2.2.2. 

Application of appropriate assessment factors 

Derivation of reference values such as a DNEL requires the selection of AFs. AFs are numerical 
values. They are used to address the differences between the experimental data and the human 
situation,  considering  the  uncertainties  in  the  extrapolation  procedure  and  in  the  available 
dataset. In principle, all data on a relevant chemical species need to be reviewed thoroughly in 
order  to  use,  as  far  as  possible,  substance-specific  information  for  the  establishment  of 
appropriate  values  for  the  various  AFs.  When  substance-specific  information  is  not  available, 
data on analogues,  which act with the same  mode of action  as the  relevant chemical  species 
under consideration, should be considered. However, when the available data do not allow the 
derivation of substance-specific or analogue-specific AFs, default AFs should be applied. Although 
it is very often necessary to rely upon them, default AFs represent a fallback position rather than 
a starting point. 

 
 
 
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99 

The setting of the overall AF is a critical step, which considers, but is not limited to: 

• 

• 

interspecies differences in toxicokinetics; 

interspecies differences in toxicodynamics; 

•  human variability in toxicokinetics; 

•  human variability in toxicodynamics. 

Interspecies  differences.  Data  from  animal  studies  are  a  typical  starting  point  for  risk 
characterisations;  thus,  differences  in  sensitivity  between  experimental  animals  and  humans 
need  to  be  addressed,  with  the  default  assumption  that  humans  are  more  sensitive  than 
experimental  animals.  Where  human  data  are  used  as  the  starting  point  for  the  risk 
characterisation,  no  extrapolation  and  no  AF  is  necessary  for  interspecies  differences  in 
sensitivity. 

Interspecies differences result from variation in the sensitivity of species due to differences in 
toxicokinetics and toxicodynamics. Some of the TK differences can be explained by differences 
in  body  size  (and  related  differences  in  basal  metabolic  rate).  Information  on  interspecies 
differences  may  be  gathered  from  the  toxicological  database  of  a  substance  or  by  using 
physiologically  based  pharmacokinetic  (PBPK)  modelling.  If  no  substance-specific  data  are 
available,  the  standard  procedure  for  threshold  effects,  used  as  a  default,  is  to  correct  for 
differences in metabolic rate (allometric scaling) and apply an additional factor of 2.5 for other 
interspecies  differences,  that  is,  TK  differences  not  related  to  metabolic  rate  (small  part)  and 
toxicodynamic  differences  (larger  part).  In  cases  where  substance-specific  information  shows 
specific  susceptibility  differences  between  species  that  are  not  related  to  differences  in  basal 
metabolic  rate,  the  additional  factor  of  2.5  for  ‘remaining  differences’  should  be  modified 
accordingly. 

Allometric  scaling  extrapolates  doses  according  to  an  overall  assumption  that  equitoxic  doses 
(when expressed in mg/kg body weight per day) scale with body weight to the power of 0.75. 
This results in different default allometric scaling factors for the different animal species when 
compared with humans (see Table 11). 

Table 11. Allometric scaling factors for different animal species when compared with humans 
(assuming the human body weight is 70 kg) 

Species 

Rat 

Mouse 

Hamster 

Guinea pig 

Rabbit 

Dog 

Body weight (kg) 

Allometric scaling factor 

0.250 

0.03 

0.11 

0.8 

2 

18 

4 

7 

5 

3 

2.4 

1.4 

Allometric  scaling  is  based  on  the  assumption,  originally  predicted  mathematically  and 
subsequently substantiated by empirical investigations (Schneider et al., 2004), that the effects 
of  toxicological  relevance  are  driven  by  the  basal  metabolic  rate,  as  it  affects  physiological 
processes such as cardiac output, blood flow and perfusion of liver and kidneys, which, in turn, 
affect the elimination/clearance of most chemicals. 

 
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Allometric scaling is an empirical approach for interspecies extrapolation of a significant number 
of kinetic processes related to toxicity. It is generally applicable to substances that are essentially 
renally excreted, but not to compounds that are highly extracted by the liver and excreted in 
the  bile.  It  appears  that  species  differences  in  biliary  excretion  and  glucuronidation  are 
independent of caloric demand (Walton et al., 2001). 

Allometric scaling according to caloric demand applies most appropriately to those substances 
for  which  the not-metabolised  parent  or  a  stable  metabolite  is  the  relevant  toxic  species  and 
clearance is according to first-order processes. Conversely, the applicability of allometric scaling 
is  less  well  supported  when  toxicity  is  a  consequence  of  exposure  to  a  very  reactive  parent 
compound (or metabolite) that is not removed from the site of formation. 

Allometric scaling should not be applied if the  effects are not dependent on metabolic  rate  or 
systemic absorption, for example in the case of local effects. Generally, as long as route-to-route 
extrapolation is not needed, allometric scaling should also not be applied in cases where doses 
in experimental animal studies are expressed as concentrations (e.g. in mg/m3 in air, ppm in 
diet, or mg/l in drinking water). The concentrations are assumed to be already scaled according 
to the allometric principle, since ventilation rate and food intake directly depend on the basal 
metabolic rate. However, once the concentration (e.g. ppm in diet) has been converted into a 
dose  (e.g.  mg/kg/day),  an  allometric  scaling  factor  has  to  be  used.  Thus,  it  is  the  dose  unit 
(original or transformed), and not the (experimental) route of application, that triggers the need 
for a species-specific factor for allometric scaling. 

For systemic effects in cases where allometric scaling is not applicable, AFs established on the 
basis  of  substance-specific  information  should  be  well  justified  and  used  in  a  case-by  case 
manner. 

4.2.2.3. 

Intraspecies differences 

Humans differ in sensitivity to toxic insult due to a multitude of biological factors, such as genetic 
polymorphism affecting, for example, toxicokinetics/metabolism, age, gender, health status and 
nutritional status. These differences can be the result of genetic and/or environmental influences. 
This  intraspecies  variation  is  greater  in  humans  than  in  the  more  inbred  experimental  animal 
population. 

It is recognised that in order to always account for the most sensitive person exposed to any 
chemical, a very large default AF would be required. That is of course not workable, and it is 
usually assumed that a default AF of 10 is sufficient to protect the larger part of the population, 
including  children  and  the  elderly.  For  threshold  effects,  this  factor  of  10  is  the  standard 
procedure, used as a default, when assessing exposure to the general population. It is recognised 
that there are differences between children and adults in toxicokinetics (especially babies in their 
first  months)  and  toxicodynamics  (especially  at  different  stages  of  development).  These 
differences  may  render  children  susceptible  to  the  toxic  effects  of  a  substance.  A  higher 
intraspecies AF for children (US EPA ( 2009) recommends from 10 up to 100 when assessing 
pesticides in relation to food safety) should be considered when the following two criteria are 
fulfilled: 

• 

• 

there  are  indications,  obtained  from,  for  example,  experiments  in  adult  animals, 
epidemiological studies, in vitro experiments and/or SARs, of effects on organ systems and 
functions that are particularly vulnerable under development and maturation in early life (in 
particular the nervous, reproductive, endocrine and immune systems, and also the metabolic 
pathways); 

there are deficiencies in the database on such effects in young animals. 

 
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This line of reasoning and these criteria of course also apply to the unborn child, that is, to the 
pregnant woman. 

For additional information on allometric scaling and its use, please refer to ECHA’s Guidance on 
Information Requirements and Chemical Safety Assessment – Chapter R.8: Characterisation of 
dose [concentration]–response for human health, Section R.8.4.3.1 (69). 

In addition to uncertainties in interspecies differences and intraspecies variability, additional AFs 
for the following elements should be considered. 

•  The nature and severity of the effect. 

•  Deviations between the exposure in the study providing the NOAEL and the estimated human 

exposure (e.g. lifetime for exposure to drinking water). 

•  Duration extrapolation. AFs for duration extrapolation should be handled on a case-by-case 
basis, to use the best available data in derivation of reference values. It is specifically noted 
that the possibility of duration extrapolation cannot be used in justifying a study waiver. 

o  subchronic to chronic: AF of 2. 

o  Subacute to chronic: an AF of 6.  

•  Dose–response relationship: 

o  extrapolation from LOAEL to NOAEL; 

o 

the slope of the dose–response curve. 

•  The overall quality of the toxicity data package. 

If the severity of the critical effect at the LOAEL (even if a NOAEL has been identified) is judged 
to be of particular significance, an additional AF might be considered necessary. Based on expert 
judgement, this AF has ranged from 2 to 10. Quantification should be determined on a case-by-
case basis taking into account the dose–response data. 

If the derivation of the DNEL was based on a LOAEL and not a NOAEL, an additional AF has to 
be considered. This factor will vary depending on the slope of the dose–response curve and the 
magnitude  of  the  effect  at  the  LOAEL.  This  extrapolation  step  should  be  based  on  expert 
judgement. The BMD concept can also be used when data allows and if it is deemed appropriate. 
Guidance  for  using  the  BMD  approach  can  be  found  in  Guidance  on  the  Biocidal  Products 
Regulation –  Volume III  human  health –  Assessment  &  evaluation 
(Parts B+C), 
Section 2.3.2.1 ( 70). The use of LOAELs to set DNELs should be a last resort; however, where 
the effects at the LOAEL are of moderate magnitude and not severe, the use of a LOAEL and an 
appropriate AF reduces the need for additional animal studies. 

To  account  for  uncertainties  related  to  data  used  to  identify  the  accumulation  potential, 
additional  AFs  may  need  to  be  applied  (see  Section 6.5.1  of  DWD  Guidance  Volume I).  In 

(69)  https://echa.europa.eu/documents/10162/17224/information_requirements_r8_en.pdf/e153243a-03f0-44c5-

8808-88af66223258?t=1353935239897. 

(70)

https://echa.europa.eu/documents/10162/2324906/biocides_guidance_human_health_ra_iii_part_bc_en.pdf/

30d53d7d-9723-7db4-357a-ca68739f5094?t=1512979002065. 

 
 
 
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addition, when available, data from PBPK modelling can be used for the purpose of refining the 
AFs.  PBPK  models  will  not  remove  all  the  uncertainty  from  the  risk  assessment  process.  The 
rationale  for  using  PBPK  models  in  risk  assessment  is  that  they  provide  a  documentable, 
scientifically  defensible  means  of  bridging  the  gap  between  animal  bioassays  and  human  risk 
estimates.  Guidance  on  the  use  of  PBPK  modelling  is  available  from  the  IPCS  project  on  the 
harmonization of approaches to the assessment of risk from exposure to chemicals, and should 
be followed (IPCS, 2010). 

4.2.3.  Dose–response assessment 

The quantitative extrapolation of hazard from animal experiments to exposed humans is based 
on the most relevant end points. In most cases, these end points should correspond to relevant 
dose  descriptors  (e.g.  BMD,  NOAEL,  NOAEC,  LOAEL,  LOAEC).  For  more  details  on  the 
identification and modification of dose descriptors for systemic toxicity, please refer to ECHA’s 
Guidance  on  Information  Requirements  and  Chemical  Safety  Assessment –  Chapter R.8: 
Characterisation of dose [concentration]–response for human health, Section R.8.4.3.1 ( 71), and 
EFSA’s 2022 guidance on the use of the benchmark dose approach in risk assessment (72). 

4.3.  Non-threshold effects 

For substances migrating into the drinking water exerting genotoxic, carcinogenic or endocrine 
disrupting effects, non-threshold mode of action is assumed, unless a threshold mode of action 
is demonstrated. For non-threshold effects, a conservative MTCtap of 0.1 µg/l should be applied 
(see Section 6.2.3). 

(71)  https://echa.europa.eu/documents/10162/17224/information_requirements_r8_en.pdf/e153243a-03f0-44c5-

8808-88af66223258?t=1353935239897. 

(72)  https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2022.7584. 

 
 
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5.  Exposure assessment 

Exposure is a quantitative estimate of the dose/concentration of a substance that humans are 
exposed to. Routes  of  exposure to humans are  oral, inhalation and dermal. In the context of 
water  intended  for  human  consumption,  the  route  of  (chronic)  exposure,  likely  to  cause  the 
highest exposure to humans, is the oral one (i.e. by direct ingestion of drinking water from the 
tap or indirectly via beverages or food, etc.). In fact, compared with the oral route, other routes 
of exposure via domestic water (e.g. dermal, when taking a shower or bath) are more acute in 
nature and considered less important here. To estimate exposure via drinking water to relevant 
chemical  species  originating  from  drinking  water  contact  materials  (DWCMs),  accurate 
quantification of the concentrations of the relevant chemical species migrating from and released 
by DWCMs into drinking water is essential. 

This  section  develops  the  concept  of  assessing  risks  related  to  substances  migrated  from 
DWCMs.  In practice, this means how to assess data  from physical testing introduced in  DWD 
Guidance Volume I with the aim of establishing the Ctap. This value, which is strictly linked to the 
exposure  (dose)  to  which  humans  are  potentially  exposed,  is  then  used  to  assess  whether  a 
starting  substance,  composition  or  constituent  fulfils  the  criteria  of  risk  acceptance  (see 
Chapter 6). 

5.1.  Determination of concentration at the tap from physical testing 

The  value  of  Ctap  compared  with  the  limit  concentration  MTCtap  must  obtained  for  relevant 
chemical species in each material type by physical testing in the worst foreseeable conditions of 
use, as instructed in Chapter 5 of DWD Guidance Volume I. The steps are as follows. 

•  Step 1.  Determine  the  material  type.  Determine  whether  the  starting  substance, 
composition or constituent is used in (and thus belongs to) organic, metallic, cementitious 
or enamel, ceramic and other inorganic material. Once the material type is determined, the 
instructions in step 2 must be followed to determine the Ctap. 

•  Step 2. If the starting substance, composition or constituent is used in a bulk material, the 
instructions  below  for  that  particular  material  type  must  be  followed.  If  the  starting 
substance,  composition  or  constituent  is  used  in  a  surface  layer  (e.g.  coating,  plating  or 
lining), the instructions below for surface layers must be followed. 

5.1.1.  Organic materials 

All testing, including preparation of the test sample and test procedures, must be conducted in 
the worst foreseeable conditions of use, as instructed in DWD Guidance Volume I, Section 5.1.2. 
All  analyses  must  be  conducted  in  accordance  with  the  analytical  quality  criteria  described  in 
DWD Guidance Volume I, Section 5.1.7. 

The general procedure  to determine the Ctap from the physical migration measurements is as 
follows: 

• 

• 

calculate the migration rate, M, of a substance from its measured concentration(s), ci, in the 
migration  water  of  a  specified  migration  period  (3rd  or  7th),  as  instructed  in  European 
standard EN 12873-1; 

calculate the Ctap using the migration rate as determined above and a conversion factor using 
(1) the default worst-case conversion factor  of 20 (e.g. no limitation  on use  for a specific 

 
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product type) or (2) a  product-specific conversion factor (73) (< 20) if  the applicant would 
like to limit the approval to necessary use of certain product(s). 

The migration periods chosen for the calculation of migration rate should be as follows. 

•  Cold  water.  The  Ctap  value  is  calculated  from  the  3rd  migration  period  obtained  from  a 
physical  migration  test  in  accordance  with  EN 12873-1  in  cold  chlorinated  and  non-
chlorinated  water.  Testing  in  two  waters  is  needed  to  demonstrate  the  worst  foreseeable 
conditions  because  these  may  vary  (see  also  Table 2,  Section 5.1.2  in  DWD  Guidance 
Volume I). 

•  Warm or  hot water.  The Ctap value is calculated  from the 7th migration period  obtained 
from a physical migration test in accordance with EN 12873-1 in warm or hot non-chlorinated 
water. Additionally, if the test piece containing the applied-for entry is a multilayer material 
and  the  applied-for  entry  is  not  contained  in  the  surface  layer,  an  extended  test  in  high 
temperature is necessary, and the Ctap value is calculated from the 22nd migration period. 

If testing in accordance with EN 12873-1 is not possible and this is demonstrated by a written 
scientific  justification,  use  of  modelling  can  be  considered.  Ctap  using  modelling  is  calculated 
similarly  to  that  from  physical  testing  by  using  the  calculated  results  instead  of  physical  test 
results. 

5.1.2.  Metallic materials 

All testing, including preparation of the test piece and testing procedures, must be conducted in 
the worst foreseeable conditions of use, as instructed in DWD Guidance Volume I, Section 5.1.2. 
All  analyses  must  be  conducted  in  accordance  with  the  analytical  quality  criteria  described  in 
DWD Guidance Volume I, Section 5.1.7. 

In the case of metallic compositions, exposure assessment is necessary for compositions that 
are tested in accordance with EN 15664-1 and are intended for use in product groups A, B or C. 
Compositions that are tested and accepted in accordance with EN 16056 according to the criteria 
for passive compositions or that are intended for use in product group D have negligible releases, 
and thus insignificant exposure is assumed. 

5.1.2.1. 

Determination of concentration at the tap values from physical 
testing in accordance with EN 15664-1 

The concentration value(s) at the tap for metallic compositions used to compare concentrations 
in test water against the corresponding MTCtap value are determined by the following. 

•  The equivalent pipe concentration, c*

EP,n(T,t), as defined in EN 15664-1. For pipe samples, 
c*
EP,n(T,t) equals the concentration of metals in water. For pipe pieces (i.e. test pieces in the 
standard), it is formed by multiplying the concentration of metals in water by VS/VTP, which 
are sample volume from the rig and volume of water in the test pieces. It corrects for the 
decreased surface area from full pipe. 

(73)  The  conversion  factors  typical  for  different  product  groups  are  reported  in  Table 5  of  Annex I  to  Commission 

Implementing Decision (EU) 2024/368. 

 
 
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•  The mean of the equivalent pipe concentrations, MEPn(T), as defined in EN 15664-1. MEPn(T) 
is the arithmetic mean of the equivalent pipe concentrations of stagnation samples from test 
line n according to the sampling plan in Annex B to EN 15664-1. 

•  MEPa(T) that is the average of the MEPn(T) of the three test lines.  

•  A multiplication factor ‘a’ according to the relevant product group (see Table 8, Annex II to 

DWD Guidance Volume I). 

•  Ctap = MEPa(T) × a. 

In the expression of MEPn(T) and MEPa(T) , T is the operation period in weeks. 

If  the  metallic  composition  does  not  belong  to  an  existing  metallic  composition  category  (see 
Table 14, Annex IV to DWD Guidance Volume I and the ECHA website), the applicant must follow 
the testing instructions in Section 5.1.4.3 of DWD Guidance Volume I. Otherwise, the applicant 
must follow the instructions in Section 5.1.4.4 of DWD Guidance Volume I. 

5.1.3. 

Cementitious materials 

All testing, including preparation of the test piece and testing procedures, must be conducted in 
the worst foreseeable conditions of use, as described in DWD Guidance Volume I, Section 5.1.2. 
All  analyses  must  be  conducted  in  accordance  with  the  analytical  quality  criteria  described  in 
DWD Guidance Volume I, Section 5.1.7. 

The general procedure to determine Ctap from the physical migration measurements is equivalent 
to that for  organic materials (see above).  The migration periods  chosen for the calculation of 
migration should be as follows. 

•  Cold  water.  The  Ctap  value  is  calculated  from  the  3rd  migration  period  obtained  from  a 
physical migration test in accordance with EN 14944-3 in cold chlorinated or non-chlorinated 
water. 

•  Warm or  hot water.  The Ctap value is calculated  from the 7th migration period  obtained 
from a physical migration test in accordance with EN 14944-3 in warm or hot chlorinated or 
non-chlorinated water. 

5.1.4. 

Enamel, ceramic and other inorganic materials 

All testing, including preparation of test piece and testing procedures, must be conducted in the 
worst-known  conditions  of  use,  as  described  in  DWD  Guidance  Volume I,  Section 5.1.2.  All 
analyses must be conducted in accordance with the analytical quality criteria described in DWD 
Guidance Volume I, Section 5.1.7. 

The  general  procedure  to  determine  the  Ctap  from  the  physical  migration  measurements  is 
equivalent  to  that  for  organic  materials  (see  above).  The  migration  periods  chosen  for  the 
calculation of migration rate should be as follows. 

•  Cold  water.  The  Ctap  value  is  calculated  from  the  3rd  migration  period  obtained  from  a 

physical migration test in accordance with EN 12873-1 in cold demineralised water. 

•  Warm or hot water. The Ctap value calculated from the 7th migration period obtained from 
a physical migration test in accordance with EN 12873-1 in warm or hot demineralised water. 

 
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5.1.5. 

Surface layers 

Guidance on the DWD: Volume II 
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Surface layers, used here to refer to coatings and platings, generally refer to any layer of material 
applied  on  a  bulk  substrate  that  differs  from  the  bulk  in  its  properties.  Coatings  and  platings 
include  organic,  metallic,  cementitious,  and  enamel,  ceramic  and  other  inorganic  materials 
applied  by  any  existing  method,  for  example  spraying,  brushing,  hot-dipping  and  vacuum 
deposition. 

All testing, including preparation of the test sample and test procedures, must be conducted in 
the worst foreseeable conditions of use, as described in DWD Guidance Volume I, Section 5.1.2, 
depending  on  the  coating  or  plating  material  in  question.  All  analyses  must  be  conducted  in 
accordance  with  the  analytical  quality  criteria  described  in  DWD  Guidance  Volume I, 
Section 5.1.7. 

The  value  of  Ctap  must  be  obtained  for  relevant  chemical  species  in  coatings  and  linings  by 
physical testing in the worst foreseeable conditions of use, as described earlier in this chapter. 
The testing should follow the criteria for the group of materials to which the type of the coating 
belongs.  For  metallic  platings  on  metallic  substrate  materials,  a  long-term  migration  test  in 
accordance  with  EN 16058  or  EN 15664-1  can  be  used  where  appropriate,  taking  into 
consideration the shape of the test piece. If EN 16058 is used, three products should be tested. 
Generally, the presentation of results must be as for EN 15664-1. 

Deviations from bulk material testing include the following. 

•  The details of the substrate material must be reported. 

•  Migration from the substrate material, for example permeability, must be assessed. This may 
be  accomplished  by,  for  example  (1)  a  screening  analysis  and/or  targeted  analysis, 
potentially followed by a targeted analysis of the migration water if the substrate material 
does  not  contain  substances  similar  to  the  surface  layer  or  (2)  a  migration  test  with  the 
intended substrate and an additional migration test with a substrate that does not contain 
any substances similar to the surface layer, followed by an analysis as in (1), and comparison 
of the results to estimate potential migration through the surface layer from the substrate. 
Other methods may be used and their suitability should be scientifically justified. 

•  Surface layer (coating, plating, lining) thickness, permeability and processing details must 

be reported. 

•  For  cementitious  coatings  (linings),  a  porosity  test  must  be  conducted  in  accordance  with 

EN 14944-3. 

•  For platings formed using a galvanic bath, there may be non-intended residual substances 
from  the  plating  bath  incorporated  into  the  plating.  A  migration  test  in  accordance  with 
EN 12873-1  should  be  performed  to  demonstrate  the  absence  of  such  substances.  The 
normal  test  procedures,  including  flushing  and  prewashing,  apply,  as  the  plated  products 
would  normally  undergo  similar  operations  after  plating.  Certain  platings  may  contain 
residual substances or compounds incorporated during plating process that may be gradually 
removed,  for  example  via  evaporation  or  other  natural  processes;  thus,  plating  may  take 
some  time  after  processing  before  reaching  its  final  state.  The  intention  of  this  test  is  to 
ensure the absence of more stable residues that may represent a risk to the quality of water 
intended for human consumption. 

 
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107 

5.2.  Determination of concentration at the tap from modelling for 

organic materials 

Determination of the Ctap from modelling in accordance with CEN/TR 16364 follows, in principle, 
the instructions laid out in Section 5.3.1 of DWD Guidance Volume I for starting substances, for 
example determination of Ctap from simulated 3rd or 7th migration periods. 

When  calculating  a  simulated  Ctap,  reasonable  worst-case  values  for  parameters  representing 
the interaction between substance, material and water must be used, for example: 

•  diffusion coefficient; 

•  partition coefficient of the substance between organic material and water (KP,W); 

• 

initial substance concentration in the final material. 

The boundaries of the applicability domain of the modelling (e.g. applies only to organic material 
where diffusion and partitioning can be estimated for starting substances), policy requirements 
for its use (e.g. physical testing is not reliable due to analytical limit of quantification challenges) 
and estimation of reasonable worst-case values of the parameters needed for the modelling are 
reported in CEN/TR 16364 and summarised in Section 5.3.1 of DWD Guidance Volume I. 

 
 
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6.  Risk acceptance 

6.1.  Main principles 

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6.1.1. 

Approaches to maximum tolerable concentration at the tap adoption 
for relevant chemical species 

The starting substance, composition or constituent can be considered to pose an acceptable risk 
if  the  Ctap  is  lower  than  the  corresponding  MTCtap  for  each  of  the  relevant  chemical  species 
associated with the specified use. 

The estimation of MTCtap values for relevant chemical species may follow one of the following 
approaches, depending on the situation. 

•  Limited  approach.  A  limited  acceptance  approach  based  on  Section 1  of  Annex VI  to 

Commission Implementing Decision (EU) 2024/365 may be followed. 

o  Limited  approach 1.  A predetermined MTCtap value  can be adopted  on the basis of  a 
robust past assessment of the toxicological properties of the relevant chemical species. 
No toxicological information should be generated for the purposes of the DWD application, 
unless  new  or  updated  information  has  emerged  since  that  past  assessment.  This 
approach  does  not  depend  on  the  migration  tier  of  the  relevant  chemical  species 
(Section 6.2.1). 

o  Limited approach 2. The identification of a hazard with a non-threshold mode of action 
results  in  a  default  stringent  MTCtap  value  of  0.1 μg/l,  in  some  cases  together  with 
additional conditions on the use of the applied-for entry (see Sections 6.2.2 and 6.2.3). 

o  Limited  approach 3.  The  available  information  is  insufficient  to  exclude  genotoxicity, 

thus resulting in a default stringent MTCtap value of 0.1 μg/l (see Section 6.2.4). 

o  Limited  approach 4.  The  available  information  for  the  relevant  chemical  species  is 
sufficient  to  exclude  genotoxicity  but  does  not  allow  a  conclusion  on  toxic  threshold 
effects, thus resulting in a default stringent MTCtap value of 2.5 μg/l (see Section 6.2.4). 

•  Comprehensive  approach.  A  comprehensive  acceptance  methodology  that  starts  from 
toxicological information requirements linked to the migration tier of the relevant chemical 
species. It considers all toxicological information available to adopt an MTCtap value, including 
by calculation via a systemic DNEL value (see Section 6.3). 

6.1.2. 

Concentration at the tap and migration tiers 

As  set  down  in  Chapter 6  of  DWD  Guidance  Volume I,  there  are  three  migration  tiers  with 
associated  toxicological  data  information  requirements,  which  expand  with  the  increased 
migration of the substance from the DWCM: 

• 

low migration tier – Ctap < 2.5 µg/l; 

•  medium migration tier – 2.5 µg/l ≤ Ctap < 250 µg/l; 

•  high migration tier – Ctap ≥ 250 µg/l. 

 
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109 

These migration tiers originate from the migration tiers used by EFSA in its guidance on preparing 
an  application  for  the  safety  assessment  of  a  substance  to  be  used  in  plastic  food  contact 
materials (FCMs) ( 74), taking into account the requirements of Annex V to the DWD. Annex V to 
the  DWD  prescribes  that  the  MTCtap  be  based  on  a  specific  migration  limit  (SML)  set  in 
Commission Regulation (EU) No 10/2011 by considering a 10 % ALF and water consumption of 
2 l per day. Numerically, the division of the boundaries of the EFSA migration tiers by 20 has 
been used to derive the DWD migration tier thresholds. The equivalent migration tiers for FCMs 
and DWCMs is shown in Table 12. 

Table 12. Migration tiers equivalents for FCM and DWCM assessments 

Migration tier 

FCM migration tier 

DWCM migration tier 

Low 

< 0.05 mg/kg food 

Ctap < 2.5 µg/l 

Medium 

0.05–5 mg/kg food 

2.5 µg/l ≤ Ctap < 250 µg/l 

High 

5–60 mg/kg food 

Ctap ≥ 250 µg/l 

It should be noted that the Ctap is determined as instructed, for example the 3rd migration period 
for organic materials in cold water. For metals, the migration tier for toxicological analysis should 
be taken as the mean of MEPa(T) for T = 16, 21 and 26 weeks for the relevant chemical species 
in a rig test in accordance with EN 15664-1, resulting from the worst test water where possible. 

6.2.  Limited approach to the adoption of a maximum tolerable 

concentration at the tap value 

6.2.1. 

Limited approach 1: adoption of a maximum tolerable concentration 
at the tap value on the basis of a robust past assessment 

Section 1.3  of  Annex VI  to  Commission  Implementing  Decision  (EU)  2024/365  outlines  cases 
where an MTCtap value for a relevant chemical species can be generated from available data and 
the  applicant  has  no  obligation  to  supply  toxicological  information,  unless  there  are  new  or 
updated data that need to be taken into consideration in setting an updated MTCtap value. Such 
cases  of  limited  acceptance  include  relevant  chemical  species  for  which  the  following  is 
applicable. 

•  A  parametric  value  is  set  under  Annex I  to  the  DWD,  in  which  case  the  MTCtap  value  is 
calculated  by  application  of  an  appropriate  ALF  to  consider  potential  multiple  routes  of 
exposure to the relevant chemical species, besides migration from products in contact with 
drinking  water.  That  MTCtap  value  is  to  be  used  for  the  purposes  of  risk  acceptance.  For 
metals, those values will be available in Annex V to Commission Implementing Decision (EU) 
2024/367;  for  certain  substances  listed  in  Annex I  to  the  DWD,  their  MTCtap  value  will  be 
found  in  the  corresponding  entry  of  the  relevant  EUPL  in  the  annexes  to  Commission 
Implementing Decision (EU) 2024/367. 

•  An  MTCtap  value  for  the  relevant  material  type  is  set  under  the  corresponding  annex  to 
Commission  Implementing  Decision  (EU)  2024/367  following  a  decision  by  the  European 
Commission  on  a  DWD  application  for  a  starting  substance,  composition  or  organic 
cementitious constituent that has been previously submitted to ECHA. That MTCtap value may 

(74)  https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2008.21r. 

 
 
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be used for the purposes of risk acceptance, unless information not included in the application 
underlying  the  earlier  European  Commission  decision  materially  impacts  upon  that  MTCtap 
value, thus requiring an updated, more appropriate value to be generated. 

•  An  authorisation  as  an  active  substance  has  been  granted  under  Regulation  (EU) 
No 528/2012 (the biocidal products regulation) on the basis of an agency opinion setting a 
defined safety threshold for the oral route and used as such for material in contact with water 
under product type 6. In such cases, that safety threshold is to be used for the purpose of 
generating an MTCtap value in accordance with Section 4.2.2. Biocidal active substances may 
be  used  with  the  intention  to  exert  such  activity  in  organic  and  cementitious  materials. 
However, only biocidal active substances of product type 6 (preservatives for products during 
storage),  in  accordance  with  Regulation  (EU)  No 528/2012,  may  be  used  as  starting 
substances or organic cementitious constituents. 

•  An SML for the relevant chemical species that was set in Regulation (EU) No 10/2011 less 
than 15 years in advance of the date of submission of the DWD application exists. In that 
case,  that  SML  divided  by  20  and  expressed  in  μg/l  is  to  be  used  as  the  MTCtap  for  the 
purposes of risk acceptance. Applicants are reminded that an SML is tied to a FCM migration 
tier and the migration tiers for the same relevant chemical species migrating from FCM and 
DWCM  may  differ.  If  the  SML  is  based  on  an  FCM  migration  tier  that  is  lower  than  the 
applicable tier for DWCM, the MTCtap value calculated from the existing SML value may be 
lower than the established Ctap value in drinking water. Acceptable risk from the use of the 
applied-for entry can thus not be demonstrated. In such cases, only through the provision of 
toxicological  information  for  the  corresponding  DWCM  migration  tier  that  allows  the 
derivation of a higher, more appropriate MTCtap value for DWD purposes can risks be shown 
to be acceptable. In addition, any additional toxicological information that exists (e.g. a new 
study that could not be considered when establishing an earlier SML) and can lead to a more 
protective  MTCtap  value  than  what  can  be  calculated  from  an  existing  SML  must  also  be 
considered by the applicant. 

Table 13  summarises  sources  of  existing  MTCtap  values  that  applicants  may  rely  upon  in  their 
applications. It is important to note that it is up to the applicants to ensure that any existing 
MTCtap value (or parametric value) is based on the latest available toxicological information for 
the relevant chemical species in question. 

Table 13. Sources of existing MTCtap values for different material types 

Material 
type 

Organic 

in 

Table 
Commission 
Implementing  Decision 
(EU) 2024/367 
Table 1 of Annex I 

Description 

For certain starting substances (e.g. acrylamide, benzene, 
vinyl chloride), the table includes MTCtap values that have 
been based on a parametric value shown in Annex I to the 
DWD or an SML value, and for which no new or updated 
data have become available since their last review. 

The table will ultimately provide MTCtap values for starting 
substances and other relevant chemical species that have 
already been reviewed by RAC, and for which no new or 
updated  data  have  become  available  since  their  last 
review. 

 
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111 

Material 
type 

in 

Table 
Commission 
Implementing  Decision 
(EU) 2024/367 
Table 1 of Annex V 

Table 3 of Annex I 

Table 4 of Annex I 

Metallic 

Table 1 of Annex V 

Cementitious  Table 3 of Annex III 

Table 1 of Annex V 

Enamel, etc. 

Table 1 of Annex V 

Table 1 of Annex V or 
Table 1 of Annex I 

Description 

For  certain  metals 
to  metallic  starting 
relevant 
substances,  the  table  includes  MTCtap  values  that  are 
presented  in  Annex V  to  the  decision  and  based  on  a 
parametric value in Annex I to the DWD or an SML value 
from  Annexes I  and  II  to  Regulation  (EU)  No 10/2011), 
and  for  which  no  new  or  updated  data  have  become 
available since their last review. 

The  table  provides  total  MTCtap  values  for  relevant 
chemical  species  that  may  arise  from  multiple  starting 
substances. 

The table provides MTCtap values for degradation products 
of  starting  substances  with  alkylphenol  structural 
moieties. 

The table includes metallic elements for which an MTCtap 
is available and for which no new or updated data have 
become available since their last review. 

For  certain  organic  cementitious  constituents  (e.g. 
acrylamide,  benzene,  vinyl  chloride),  the  table  already 
includes  MTCtap  values  that  have  been  based  on  a 
parametric value in Annex I to the DWD, and for which no 
new  or  updated  data  have  become  available  since  their 
last review. 

The table will ultimately provide MTCtap values for organic 
cementitious  constituents  and  other  relevant  chemical 
species  that  have  already  been  reviewed  by  ECHA’s 
Committee for Risk Assessment, and for which no new or 
updated  data  have  become  available  since  their  last 
review. 

For  certain  metals  relevant  to  organic  cementitious 
constituents,  an  MTCtap  is  available  on  the  basis  of  a 
parametric value in Annex I to the DWD or an SML value 
from Annexes I and II of Regulation (EU) No 10/2011. 

For  certain  elements,  the  table  already  includes  MTCtap 
values  that  have  been  based  on  the  corresponding 
existing parametric value shown in Annex I to the DWD, 
and  for  which  no  new  or  updated  data  have  become 
available since their last review. 

For  certain  organic  impurities  (e.g.  polycyclic  aromatic 
hydrocarbons), the table includes MTCtap values that are 
based  on  a  parametric  value  shown  in  Annex I  to  the 
DWD, and for which no new or updated data have become 
available since their last review. 

 
 
 
 
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6.2.2. 

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Limited approach 2: maximum tolerable concentration at the tap 
value for relevant chemical species classified as carcinogenic, 
mutagenic or toxic to reproduction category 1A or 1B / endocrine 
disruptor human health category 1/PBT/vPvB/PMT/vPvM 

Consumers should not be exposed to substances of very high concern, particularly those of very 
high concern for human health, as a result of the use of starting substances, compositions and 
constituents in the manufacture of DWCMs. In the context of DWCM, a relevant chemical species 
that is a starting substance, an organic cementitious constituent, a substance constituent or a 
non-intentionally added species is considered to be of very high concern if either of the following 
apply: 

• 

• 

it has the hazard classification, whether harmonised or self-classification, of (1) carcinogenic, 
mutagenic or toxic to reproduction (CMR) category 1A or 1B, (2) ED human health category 
1,  (3)  persistent,  bioaccumulative  and  toxic  (PBT),  (4)  very  persistent  and  very 
bioaccumulative  (vPvB),  (5)  persistent,  mobile  and  toxic  (PMT)  or  (6)  very  persistent  and 
very mobile (vPvM) established under Regulation (EU) No 1272/2008; 

it is identified as a substance of very high concern under the candidate list established under 
Article 59  of  Regulation  (EC)  No 1907/2006,  except  those  identified  on  the  basis  of 
Article 57(f) of Regulation (EC) No 1907/2006 only for the environment. 

Starting substances, compositions or constituents that themselves fall under one of the above 
classes or the use of which gives rise to migration of relevant chemical species that fall under 
one of the above classes should not be used as far as possible in the manufacture of DWCM. 

Nevertheless, if an application involves a relevant chemical species that falls under one or more 
of the above classes, the approval of the applied-for entry into the EUPL is only possible if a very 
stringent migration limit applies to the relevant chemical species. Moreover, the concentration 
of the relevant chemical species in the final material should be restricted. 

The rationale behind this approach is that: 

•  all  consumers,  including  vulnerable  groups,  are  regularly  and  continuously  exposed  to 

substances migrating from DWCMs; 

•  products in contact with drinking water have a long-lasting article service life, with difficulty 

in guaranteeing low migration from them after several years (due to ageing effects); 

• 

• 

• 

there is analytical uncertainty around the very low migration limit potentially applicable to 
the CMR/ED/PBT/vPvB substances; 

the  approach  is  in  line  with  chemicals  strategy  for  sustainability,  which  shapes  the  future 
landscape in chemical risk management in the EU; the strategy discusses a generic restriction 
on CMR substances in several article types, including FCM; 

the  use  of  CMR,  ED  and  PBT/vPvB  substances  can  also  be  largely  avoided  in  all  life-cycle 
stages, including the production and waste stage (which are not covered by the DWD); 

•  PBT/vPvB/PMT/vPvM substances can always be considered relevant for posing risks to human 
health due to the lifetime direct exposure of humans to such substances as a result of their 
migration from DWCM, but also for the following additional reasons. 

 
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113 

o  They may cause exposure via the environment, as, if present in drinking water, they will 
eventually  be  released  into  the  environment  where  they  will  persist,  accumulate  and 
contaminate the soil, ground water, etc., leading to human exposure. 

o  Due to their (very) bioaccumulative, (very) persistent and/or (very) mobile properties, 
accumulation of such substances in the environment is difficult to reverse, and the effects 
of  this  accumulation  are  often  difficult  to  predict  in  the  long  term.  Therefore,  it  is 
considered necessary to minimise emissions and exposure, since potential risks to human 
health cannot be reliably assessed and excluded. 

The reader may note that Section 6.5 of Annex I to Regulation (EC) No 1907/2006 (the REACH 
regulation) requires that: 

For  substances  satisfying  the  PBT  and  vPvB  criteria  the  manufacturer  or  importer  shall  use  the 
information  as  obtained  in  Section 5,  Step 2  when  implementing  on  its  site,  and  recommending  for 
downstream  users,  RMM  [risk  management  measures]  which  minimise  exposures  and  emissions  to 
humans and the environment, throughout the life-cycle of the substance that results from manufacture 
or identified uses. 

The  applied-for  entry  may  be  accepted  in  the  corresponding  EUPL  only  if  any  of  the  relevant 
chemical species that is of very high concern meets all the following criteria. 

• 

• 

It does not migrate at a level resulting in a Ctap value higher than 0.1 µg/l or higher than the 
corresponding MTCtap calculated from a parametric value set under Annex I to the DWD by 
application of an appropriate ALF. 

It is present in the final material at a concentration lower than 0.1 % weight per weight ratio 
(w/w). If the available analytical method does not demonstrate unequivocally that the MTCtap 
value has been met, the content of the substance in the final material must be lower than 
0.02 %  w/w;  this  lower  concentration  limit  aims  to  ensure  that  migration  is  as  low  as 
possible (75) (76). 

•  The relevant chemical species is only one of the following: 

o  a non-intentionally added species, 

o  a substance constituent, 

o  a monomer of a main polymer of the contact material. 

For such relevant chemical species, applicants generally do not need to provide any toxicological 
data.  However,  if  information  (e.g.  a  parametric  value  or  toxicological  data)  exists  and  this 
supports the derivation of an MTCtap value that is lower than 0.1 μg/l, such information must be 
considered by the applicant. Migration data and information on their concentration in the final 
material will always be required, however. 

(75)  ECHA  has  previously  investigated  the  presence  of  CMR  category 1A/1B  substances  in  childcare  articles. 
Appendix A6.1 of the report provides details of analytical methods for CMR substances that may be of relevance 
for  measuring  such  substances  in  polymeric  matrices.  The  investigation  was  published  on  31 October  2023 
(https://echa.europa.eu/completed-activities-on-restriction). 

(76)  There  is  evidence  that  a  content  lower  than  0.1 %  in  the  material  does  not  guarantee  a  migration  lower  than 
0.1 µg/l. See ‘Model calculations and specifications based on an acceptability of 0.1 µg/l of substances in tap water’, 
unpublished meeting document of the subcommittee (‘W4’) to the Dutch committee of experts, document No W4 
03-033, 2003, https://wetten.overheid.nl/BWBR0030279/2024-01-01. 

 
 
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Meeting the requirement detailed in the second point above requires the estimation of a chemical 
concentration in a solid matrix. For non-reactive relevant chemical species, a calculation-based 
estimation may be sufficient. For example, for a non-reacting additive to an organic material, its 
maximum concentration should be sufficient. For a non-reactive impurity of a starting substance 
or of an organic cementitious constituent, the estimation can be based on a simple multiplication 
of the maximum concentration of the impurity in the starting substance or organic cementitious 
constituent by the maximum dosage of the starting substance or the cementitious constituent 
in the formulation to produce the final material. 

For relevant reactive chemical species (e.g. a monomer or other reactant or a reactive additive), 
a measurement of its residual concentration in the solid matrix using an appropriate analytical 
method may be required. 

Important note. These requirements for classified relevant chemical species do not apply to 
elements  released  into  the  migration  water  from  constituents  or  impurities  of  metallic 
compositions or from constituents of enamels, ceramic or other inorganic compositions. Those 
only  need  to  respect  the  corresponding  MTCtap  value  shown  in  Annex V  to  Commission 
Implementing Decision (EU) 2024/367. Where the MTCtap value is missing from that annex or is 
not based on a parametric value in Annex I to the DWD or an SML established in Regulation (EU) 
No 10/2011,  the  applicant  must  provide  the  toxicological  information  required  under  the 
migration tier for the element established in physical migration tests and use that information 
to generate and propose in their application a new MTCtap value. 

6.2.3. 

Limited approach 3: maximum tolerable concentration at the tap 
value for relevant chemical species that are potential carcinogens, 
mutagens or endocrine disruptors with a non-threshold mode of 
action 

A relevant chemical species that is a starting substance, an organic cementitious constituent, a 
substance constituent or a non-intentionally added species and is not classified as a carcinogen, 
mutagen or ED human health category 1 according to Regulation (EC) No 1272/2008 (the CLP 
regulation)  but  which,  from  the  available  toxicological  data,  is  suspected  to  potentially  exert 
mutagenic,  carcinogenic  or  endocrine  disruptive  effects  for  human  heath  via  a  non-threshold 
mode of action, needs to comply with an MTCtap of 0.1 µg/l or an MTCtap value calculated from 
an existing parametric value. This also applies to relevant chemical species that are classified 
according to Regulation (EC) No 1272/2008 as mutagenic, carcinogenic or EDs for human health 
category 2. 

The use of substances with genotoxic properties should be avoided as much as possible. 

6.2.4. 

Limited approach 4: maximum tolerable concentration at the tap 
value for relevant chemical species for which available toxicological 
information is insufficient 

Section 1.4 of Annex VI to Commission Implementing Decision (EU) 2024/365 outlines additional 
cases where a default MTCtap value for a relevant chemical species can be adopted in the light 
of missing information, as follows. 

• 

• 

If  the  available  information  for  the  relevant  chemical  species  is  insufficient  to  exclude 
genotoxicity, an MTCtap of 0.1 µg/l applies. 

If  the  available  information  for  the  relevant  chemical  species  is  sufficient  to  exclude 
genotoxicity  but  does  not  allow  a  conclusion  on  toxic  effects  listed  under  Part 2.1.2  of 

 
Guidance on the DWD: Volume II 
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115 

Section 2 of Annex VI to Commission Implementing Decision (EU) 2024/365, an MTCtap  of 
2.5 µg/l  applies.  However,  such  a  generic  limit  cannot  be  applied  for  genotoxicity, 
carcinogenicity or endocrine disruption affecting human health with a non-threshold mode of 
action. 

More  generally,  for  every  relevant  chemical  species  for  which  toxicological  information  is 
required, provision of data on its genotoxicity should be considered the absolute minimum. 

A summary of the scenarios described above for the different types of relevant chemical species 
is provided in Table 14. 

 
116 

Table 14. Summary of limited risk acceptance approaches 

Scenario 

Risk acceptance criterion 

Guidance on the DWD: Volume II 
Version 1.0 – January 2025 

Relevant chemical species 

s
u
o

i
t
i
t
n
e
m
e
c

t
n
e
u
t
i
t
s
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c

c
i
n
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t
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t
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t
s
n
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c

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c
n
a
t
s
b
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y
l
l
a
n
o

i
t
n
e
t
n

i

d
e
d
d
a

-
n
o
N

n
o

i
t
i
s
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p
m
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C

t
n
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t
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t
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e
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u
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g
n
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t
r
a
t
S

n
o

i
t
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s
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p
m
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y
t
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r
u
p
m

i

Classified  CMR  category 1,  ED  human 
health category 1, PBT/vPvB, PMT/vPvM 

Ctap < 0.1 µg/l (or < MTCtap from a parametric value) 
and 

 

 

 

 

Concentration 
< 0.02 % w/w if analytical method is uncertain and 

final  material  < 0.1 %  w/w  or 

in 

The relevant chemical species is any of the following: 

• 

• 

• 

a non-intentionally added substance, 

a substance constituent, 

a monomer or other reactant of a main polymer in 
the material. 

Ctap < 0.1 µg/l (or < MTCtap from a parametric value) 

 

 

 

 

Ctap < MTCtap from a parametric value 

 

 

 

 

 

 

Potential  carcinogen,  mutagen  or  ED 
human  health  with 
identified  non-
threshold  mode  of  action  based  on 
available toxicological data  

Parametric  value  is  available  under 
Annex I to the DWD (and in Annex V to 
Commission 
Implementing  Decision 
(EU) 2024/367) 

MTCtap  adopted  by 
the  European 
Commission  is  available,  following  a 

Ctap < existing  MTCtap 
toxicological information is available) 

(unless  new  or  updated 

 

 

 

 

 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Guidance on the DWD: Volume II 
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117 

Scenario 

Risk acceptance criterion 

Relevant chemical species 

s
u
o

i
t
i
t
n
e
m
e
c

t
n
e
u
t
i
t
s
n
o
c

c
i
n
a
g
r
O

t
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t
i
t
s
n
o
c

e
c
n
a
t
s
b
u
S

y
l
l
a
n
o

i
t
n
e
t
n

i

d
e
d
d
a

-
n
o
N

n
o

i
t
i
s
o
p
m
o
C

t
n
e
u
t
i
t
s
n
o
c

e
c
n
a
t
s
b
u
s

g
n
i
t
r
a
t
S

n
o

i
t
i
s
o
p
m
o
C

y
t
i
r
u
p
m

i

previous DWD application to ECHA 

Safety threshold (oral) is available from 
a  biocidal  active  substance  approval 
under Regulation (EU) No 528/2012 

Ctap < MTCtap generated from the existing safety value 

 

 

Only biocidal active substances of product type 6 can be 
used in DWCM 

SML value is available in Regulation (EU) 
No 10/2011 and is less than 15 years old 
at the time of the DWD application to the 
agency 

Ctap < SML ÷ 20  (unless  new  toxicological  information 
requires  a  more  appropriate  MTCtap  value  or  in  certain 
cases where the FCM and DWCM migration tiers differ) 

 

 

 

 

 

 

The  available  information  is  insufficient 
to exclude genotoxicity 

Ctap < 0.1 µg/l 

The available information is sufficient to 
exclude genotoxicity but does not allow 
a conclusion on other toxic effects with a 
threshold mode of action 

Ctap < 2.5 µg/l 

 

 

 

 

 

 

 

 

 

 

 

 

The available information is sufficient to 
exclude  genotoxicity  but  raises  concern 
over  toxic  effects  with  a  non-threshold 
mode of action 

Ctap < 0.1 µg/l (or < MTCtap from a parametric value) 

 

 

 

 

 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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6.3.  Comprehensive approach to the adoption of a maximum tolerable 

concentration at the tap value on the basis of toxicological 
information 

6.3.1.  Maximum tolerable concentration at the tap for a relevant chemical 

species of low migration potential 

If  the  relevant  chemical  species  has  a  low  migration  potential  (Ctap < 2.5 µg/l  from  migration 
studies) and does not have a parametric value, the MTCtap will be calculated according to the 
following rules. 

• 

• 

If genotoxicity studies or other available toxicological information indicate potential toxicity 
exerted  by  non-threshold  mode  of  action  (e.g.  endocrine  disruption),  an  MTCtap  value  of 
0.1 µg/l  will  apply.  This  limit  also  applies  when  toxicological  data  are  not  available,  for 
example for a non-intentionally added species for which it is not possible to set an appropriate 
MTCtap (see Section 6.5). 

If the genotoxicity/mutagenicity studies show clearly negative results (see Section 3.4) and 
no  other  toxicity  data  that  show  hazard  that  leads  to  an  MTCtap  lower  than  2.5 µg/l  is 
available, the MTCtap is set to 2.5 µg/l. 

In  general,  when  the  toxicological  information  provided  by  the  applicant  covers  only  the 
information requirements for the low migration tier, the MTCtap cannot be higher than the upper 
boundary of the tier, that is, it cannot be higher than 2.5 µg/l. However, there may be cases 
where the applicant considers that the Ctap value is relatively close to the upper boundary of the 
tier or cases where the Ctap is uncertain due to challenges with the analytical method used. Such 
cases may raise concern that the products that are associated with the relevant chemical species 
may  fail their certification if the Ctap in the migration tests  conducted for product certification 
exceeds  2.5 µg/l.  In  such  cases,  the  applicant  may  wish  to  consider  assessing  the  standard 
toxicological  information  of  the  next  migration  tier  up  in  order  to  establish  (by  using 
equation (1)) a DNEL leading to a more appropriate MTCtap value that is higher than 2.5 µg/l. 
The  applicant  must  justify  the  provision  of  toxicological  information  that  corresponds  to  the 
information  requirements  of  the  medium  migration  tier.  Again,  if  this  approach  is  taken,  the 
MTCtap proposed (and accepted into the EUPL) cannot be higher than the upper boundary of that 
tier, that is, it cannot be higher than 250 µg/l. 

The MTCtap can be lower than the upper boundary of the tier, even in the absence of a positive 
genotoxicity study, if there is additional toxicological information that shows a toxicological effect 
with a non-threshold mode of action (e.g. endocrine disruption) or if the additional information 
supplies a DNEL that, according to equation (1), results in an MTCtap value lower than 2.5 µg/l. 
The reader is reminded that under Part 1.5 of Section 2 of Annex V to Commission Implementing 
Decision (EU) 2024/365, ‘any other relevant toxicological information that is available [beyond 
the standard information for the relevant migration tier] shall be identified and considered’. 

6.3.2.  Maximum tolerable concentration at the tap for a relevant chemical 

species of medium migration potential 

If the relevant chemical species shows a medium migration potential (Ctap is in the range of 2.5–
250 µg/l), an MTCtap can be established from the toxicological data required under this migration 
tier; in particular, it is possible to derive a dose at which no effect will occur, the DNEL, from 
repeated dose toxicity data (see Chapter 4). The MTCtap is therefore calculated according to the 
following equation: 

 
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119 

(1) MTCtap (mg/l) = 

DNEL (mg/kg/d) ×

60 (kg)

 (l/d)

2

  ×  ALF (

77
)

Essentially,  the  MTCtap  is  calculated  assuming  a  dose  below  which  no  effect  will  occur  when 
ingested by an adult (with a body weight of 60 kg) who consumes 2 l of drinking water per day, 
and  by  applying  the  so-called  ALF  (see  Section 6.4).  The  supplementary  ALF  is  introduced  to 
take into account that only a fraction of the dose of a substance may originate from the product 
in  contact  with  drinking  water  for  which  the  MTCtap  is  calculated,  as  exposure  to  the  same 
substance  may  occur  via  other  products  or  originate  from  different  sources  into  the  drinking 
water. 

In general, the MTCtap cannot be higher than the upper boundary of the tier; therefore, in this 
case, it cannot be higher than 250 µg/l. 

The MTCtap is to be set at 0.1 µg/l if there is toxicological information that shows a toxicological 
effect with a non-threshold mode of action (e.g. genotoxicity or endocrine disruption). 

6.3.3.  Maximum tolerable concentration at the tap for a relevant chemical 

species of high migration potential 

If the relevant chemical species shows a high migration potential (Ctap > 250 µg/l), the MTCtap 
will  be  calculated  using  equation (1).  Again,  the  MTCtap  is  to  be  set  at  0.1 µg/l  if  there  is 
toxicological information that shows a toxicological effect with a non-threshold mode of action 
(e.g. genotoxicity or endocrine disruption). 

For organic starting substances, there is another parameter that may play a role in setting the 
MTCtap for the high migration tier: a limit based on total organic carbon (TOC). TOC is used for 
product acceptance for organic materials in contact with drinking water; for organic materials, 
the TOC as measured in the product migration tests cannot be higher than 0.5 mg/l. Therefore, 
if the MTCtap of a starting substance is higher than the TOC limit (0.5 mg/l) ‘normalised’ to the 
carbon  content  in  the  starting  substance,  then  the  applicant  needs  to  indicate  this  in  the 
application. Consequently, the MTCtap value for the starting substance will be highlighted in the 
EUPL to enable the certification process to take this into consideration. 

In practice, this means that, for starting substances with a Ctap of > 250 µg/l, the applicant 
should: 

• 

• 

calculate the MTCtap according to equation (1); 

compare the MTCtap with the TOC limit normalised to organic carbon (= 0.5 mg/l × molecular 
weight substance / molecular weight of carbon in substance) to decide whether the MTCtap 
value should be highlighted in the EUPL. 

Note that the TOC is determined as non-purgeable organic carbon in water (in accordance with 
EN 1484). The process of TOC determination typically involves removal of inorganic carbon and 
conversion of organic carbon into CO2, which can be measured, and the amount of carbon then 
determined.  According  to  EN 1484,  suitable  methods  for  CO2  determination  include  infrared 
spectrometry,  titration,  thermal  conductivity,  conductometry  and  coulometry.  In  addition  to 
organic carbon, water typically contains inorganic carbon such as CO2 or ions of carbonic acid, 
which needs to be removed prior to TOC determination by purging with a suitable gas. If the 
substance is purgeable (e.g. benzene, toluene), it will not contribute to the TOC, and therefore 

(77)  Note that the MTCtap in the equation is expressed in mg/l, while the migration tier thresholds are expressed in μg/l. 

 
 
 
 
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the TOC criterion does not apply. Nonetheless, it is considered that the majority of TOC (~ 90 %) 
is  non-purgeable  and  methods  such  as  EN 1484  are  valid.  In  cases  where  purgeable  organic 
carbon analysis is necessary, methods exist but may have high uncertainties. 

The MTCtap calculation according to different migration tiers is summarised in Table 15. 

Table 15. MTCtap values from toxicological data according to different migration tiers under the 
comprehensive approach 

Relevant migration tier 

Equation for 
MTCtap 

Conditions and MTCtap value 

Low migration tier 
(Ctap < 2.5 µg/l) 
Medium migration tier 
(2.5 µg/l ≤ Ctap < 250 µg/l) 

Not applicable  Substance is not genotoxic  MTCtap = 2.5 µg/l 

Equation (1) 

No concern for toxicity with a non-threshold mode of 
action  MTCtap is calculated; MTCtap cannot be higher 
than 250 μg/l 

High migration tier 
(Ctap ≥ 250 µg/l) 

Equation (1) 

No concern for toxicity with a non-threshold mode of 
action  MTCtap is calculated 

If MTCtap from equation (1) exceeds 0.5 mg/l × MW / 
MW_C  MTCtap is calculated and highlighted in 
EUPL (a) 

All migration tiers 

Not applicable  Concern for toxicity with non-threshold mode of action 

 MTCtap = 0.1 µg/l 

(a) 
NB: 

Criterion valid for starting substances only where TOC is determined to be non-purgeable organic carbon. 
MW, molecular weight of a substance; MW_C, molecular weight of carbon in a substance. 

6.4.  Selection of allocation factors 

Annex V  to  Commission  Implementing  Decision  (EU)  2024/367  presents  MTCtap  values  for 
several elements and ions for which a parametric value exists in Annex I to the DWD or an SML 
appears in Regulation (EU) No 10/2011. Where a parametric value is used as the basis for the 
MTCtap, it has been multiplied by an ALF (expressed as a percentage) to exclude other possible 
sources  of  the  substance  in  the  drinking  water  from  being  taken  into  account.  The  principles 
behind  the  selection  of  the  ALF  are  presented  below  and  can  be  considered  where  applicants 
wish  to  generate  MTCtap  values  that  are  missing  from  Annex V  to  Commission  Implementing 
Decision (EU) 2024/367. 

•  The  standard  ALF  is  equal  to  10 %  for  starting  substances  and  organic  cementitious 
constituents.  It  accepts  a  maximum  contribution  from  organic  and  cementitious  DWCMs 
equal to 10 % of the maximum concentration at consumers’ taps (e.g. the parametric value 
of a substance, where that exists in Annex I to the DWD). In a few cases, where it can be 
demonstrated that the DWCM is the only source of the substance at the tap, the coefficient 
can be set to 100 %. This applies to, for example, vinyl chloride, which is used as a monomer 
for PVC polymer production, and no other source for it into drinking water is known). 

•  For elements released from metallic compositions, the standard ALF is equal to 50 % on the 
assumption that other sources of contamination are possible. An exception to this assumption 
is lead, where the only source of contamination is metallic contact materials (WHO, 2017). 
In a few cases, an ALF of 90 % can be used where metallic products in contact with drinking 
water  constitute  the  only  major  source  of  contamination  or  where  the  toxicological 
(parametric) value is substantially high (e.g. the case of copper or zinc), so that contributions 

 
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121 

from other sources into the drinking water would reliably not result in the exceedance of the 
toxicological (parametric) value. 

•  For elements released from enamel, ceramic and other inorganic materials, the standard ALF 
is also set to 10 %.  When setting up the ALF, lower (e.g. currently 5 % for cadmium and 
lead) or higher (e.g. currently 50 % for aluminium and cobalt) values may be considered. 
The  applicant  should  consider  the  toxicological  concern  and  all  sources  from  which  the 
element  may  end  up  in  drinking  water  when  proposing  a  value.  Higher  values  could 
potentially be more acceptable for low toxicity elements that do not have other sources. In 
contrast,  high  toxicity  elements  that  have  multiple  sources  from  which  to  enter  drinking 
water may be considered for lower values. Evaluation to determine a new ALF may be done 
on a case-by-case basis. 

Upon its adoption into legal text, Annex V to Commission Implementing Decision (EU) 2024/367 
does not include MTCtap values that are based on toxicological values that are not reported in EU 
legislation; as such, values originating from the work of the World Health Organization, national 
competent authorities, etc., have not been considered. Applicants may review any such available 
value  and  decide  on  and  justify  in  their  applications  its  use  as  a  basis  for  generating  a  new 
MTCtap. More importantly, applicants should justify their choice of ALF. 

6.5.  Risk acceptance methodology 

6.5.1. 

Standard methodology 

The aim of the risk assessment is to ensure that the DWCM will pose an acceptable risk to human 
health or, in other words, that migration of relevant chemical species from the DWCM to water 
will be lower than their corresponding maximum tolerable concentrations. 

This  means  that  for  each  relevant  chemical  species  the  Ctap  as  defined  in  accordance  with 
Chapter 5 needs to be lower than the MTCtap as derived in accordance with Sections 6.2 and 6.3. 
The applicant is responsible for identifying specific conditions of use if required to lower the Ctap 
below the MTCtap. 

Special care should be taken by the applicant in those cases where the Ctap as calculated from 
migration studies is very close to the MTCtap as derived according to the rules set in Sections 6.2 
and 6.3., especially when the MTCtap is set to be equal to the upper boundary of the migration 
tier (e.g.  MTCtap = 2.5 µg/l for a  relevant  chemical species at the low migration tier). In such 
cases, the applicant is advised to evaluate whether providing toxicity data relevant to the next 
tier up (medium migration, 2.5–250 µg/l) would allow the setting up of a more appropriate, but 
still protective, higher MTCtap. 

6.5.2.  Metallic compositions 

Acceptance of a metallic composition is determined according to the following steps (see also 
Annex I). 

Step 1. Determination of whether (1) the composition falls within an existing metallic material 
category  or  (2)  the  creation  of  a  new  category  is  necessary.  In  the  case  of  (2),  follow  the 
instructions for creating a new category in Section 5.1.4.3 of DWD Guidance Volume I. 

Step 2.  Determination  of  the  product  group.  For  intended  use  in  product  group A,  B  or  C, 
proceed  to  step 3.  For  intended  use  in  product  group D,  no  assessment  is  required,  as  the 
potential exposure and thus risk to human health is considered negligible. 

 
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Step 3. Determination of whether the metallic composition exhibits passivity in water. If yes, 
then  the  instructions  for  compositions  tested  in  accordance  with  EN 16056,  that  is,  passive 
compositions,  in  Section 6.5.2.2  should  be  followed.  Otherwise,  the  instructions  for  metallic 
compositions tested in accordance with EN 15664-1 in Section 6.5.2.1 should be followed. See 
also step 4. 

Step 4. If the metallic composition is used as a bulk material, follow the instructions for testing 
and  exposure  assessment  in  Sections 5.1.4  and  5.2.2  of  DWD  Guidance  Volume I  and 
Section 5.1.2 of this document, respectively. If the metallic composition is used in plating, that 
is,  in  a  surface  layer,  follow  the  instructions  for  testing  and  exposure  assessment  in 
Sections 5.1.4 and 5.2.2 of DWD Guidance Volume I and for surface layers in Section 5.1.5 of 
this document. 

6.5.2.1. 

Metallic compositions tested according to EN 15664-1 

Determine the relevant concentration values according to Section 5.1.2. Metallic materials tested 
in accordance with EN 15664-1 may be accepted by testing. 

In  the  acceptance  of  metallic  compositions,  the  criteria  considered  are  the  absolute 
concentrations of metals (criterion A) and the trends in concentration change (criterion B) as a 
function of time. 

For the assessment of the test rig results in accordance with EN 15664-1, the Ctap derived from 
the  arithmetic  mean  of  the  equivalent  pipe  concentrations  MEPa(T),  the  ‘a’  factor  (see  also 
Section 5.1.2),  and  the  arithmetic  mean  (c*
EP(T,4 hour))  of  the  4-hour  stagnation  values 
(c*

EP,n(T,4h)) are to be considered. 

The metallic composition can be accepted for a product group with the assumed contact surface 
a  (see  Table 8,  Annex II  to  DWD  Guidance  Volume I),  if  the  following  criteria  are  met  for  all 
required test waters. 

Criterion A 

The applicable MTCtap values (also refer to Annex V to Commission Implementing Decision (EU) 
2024/367) must be met for all analysed elements beginning from week 16. It is considered that 
a pipe rig test has three periods, (1) conditioning, (2) transition and (3) stability, all of which 
are  associated  with  different  metal  migration  behaviours  (Eisnor  and  Gagnon,  2003).  Risk 
acceptance should be evaluated during the stability period, as during this time potential corrosion 
product layers on the surface, etc., have formed and metal migration rates have stabilised. In 
EN 15664-1, week 16 is considered as sufficient time for this to take place. 

Criterion A is considered fulfilled if: 

•  Ctap (MEPa(T) × a) ≤ MTCtap for T = 16, 21 and 26 weeks or T = 16, 21, 39 and 52 weeks if 

the test is extended to 52 weeks due to non-compliance in criterion B. 

Criterion B 

Metal concentrations (parameters) should not increase so that there is a risk of exceeding the 
MTCtap beyond the duration of the test. 

 
 
 
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123 

Criterion B is considered fulfilled if one of the three criteria below is fulfilled: 

•  MEPa(Tb) ≥ MEPa(T) 

for {Tb, T} = {12, 16}, {16, 21} and {21, 26} {weeks}; 

•  a negative slope of a linear fit of the c*

EP(T,4-hour) for T > 12 weeks is obtained; 

• 

c*

EP(T,4 hour) × a ≤ 0.5 × MTCtap 

for T = {16–26 weeks}. 

The test may be extended up to 1 year if criterion B is not met after 26 weeks. 

If  the  test  is  extended  to  1 year,  criterion B  is  considered  fulfilled  if  one  of  the  three  criteria 
below is fulfilled: 

•  MEPa(Tb) ≥ MEPa(T) 

for {Tb, T} = {26, 39} and {39, 52} weeks; 

•  a negative slope of a linear fit of the c*

EP(T,4 hour) for T > 26 weeks is obtained; 

• 

c*

EP(T,4 hour) × a ≤ 0.5 × MTCtap 

for T = {26–52} weeks. 

For  cases  where  all  testing  and  water  analyses  were  conducted  properly,  the  assessment  of 
whether  criteria A  and  B  have  been  fulfilled  may  be  quite  straightforward.  This  could  happen 
when, for example, the Ctap values are well below the corresponding MTCtap values for all relevant 
chemical species and the visual representations of the results as curves of concentration versus 
time reveal a clearly decreasing trend. Nonetheless, when assessing acceptance, both criteria A 
and B must be numerically met according to the conditions set above. 

In other cases, compliance with these criteria can be difficult to establish clearly. This could be 
due to closeness of measured Ctap values to MTCtap values or because of deviations (outliers) in 
the test results due to uncertainties in analysis or variations in the test water composition. In 
such cases, the complete set of available data must be considered. For the test rig, according to 
EN 15664-1 these are: 

• 

results of individual test lines; 

•  4-hour stagnation results; 

•  parameters of water composition; 

• 

• 

temperature of the test rig; 

stagnation samples that were in addition to the requirements in EN 15664-1. 

In addition, for these unclear cases it is advisable to use expert assessment in interpreting the 
results. 

6.5.2.2. 

Metallic compositions tested according to EN 16056 (passivating 
alloys) 

In  cases  where  an  alloy  is  claimed  to  be  passive,  forming  a  stable  corrosion  layer  to  prevent 
general  corrosion,  this  must  be  proven  by  testing  the  stability  of  the  passive  layer  via  an 
electrochemical test in accordance with EN 16056 or equivalent. 

Sufficient  stability  is  displayed  if  the  measured  pitting  potential,  Epit,  is  higher  than  the  free 
corrosion potential + 500 mV. 

 
 
 
 
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6.5.3. 

Surface layers (coatings, platings and linings) 

Evaluation  of  surface  layers  (coatings,  platings  and  linings)  of  organic,  metallic,  cementitious 
materials or materials of inorganic origin are to be carried out as for bulk compositions. 

Due to the nature of surface layers, they have additional requirements to bulk material testing, 
which are described in Section 3.3 of DWD Guidance Volume I. In practice, a surface layer and 
its properties should be described in a sufficient manner so that an evaluation of the migration 
of substances  contained in the layer and in the substrate  can be made. For this purpose, the 
properties  that  must  be  described  may  include  layer  thickness,  adhesion,  ductility,  hardness, 
porosity, hydrogen embrittlement, fatigue and processing details. 

 
 
 
 
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8.  Annex I – Schematic of the metallics acceptance 
process 

Identification of the composition and uses: standard number, 
constituents, impurities, chemical composition and processing history 
(see Chapters 2 and 3 in DWD Guidance Volume I)

Yes

Does the alloy exhibit 
passivation due to surface 
treatment or chemical 
composition?

No

Describe whether passivity is 
due to surface treatment or 
chemical composition of bulk.
Electrochemical test in 
accordance with EN 16056

Is Epit > free corrosion 
potential +500 mV

Will the composition 
be used only in 
product group D (i.E. 
negligible water 
contact surface area)?

Yes

No

No testing 
necessary. 
Composition 
accepted

Is the composition 
used as a plating?

Yes

Yes

No

No

Composition 
accepted into passive 
category

Composition 
rejected

Does the composition 
belong to an existing 
composition 
category?

Follow the additional 
conditions for 
platings; see Section 
6.5.3

Yes

No

Addition of a composition to an 
existing category by testing In 
accordance with EN 15664-1; see 
Section 5.1.4.3 in DWD Guidance 
Volume I 

Creation of a new category by 
testing as a reference In 
accordance with EN 15664-1; see 
Section 5.1.4.4 in DWD Guidance 
Volume I

No

Provide toxicological data 
according to migration tier 
and physico-chemical 
property data, as for organics 
(see Decision 2024/365)

Do all the elements have 
an accepted existing 
MTCtap?

Yes

Does the composition 
satisfy all relevant 
acceptance criteria in 
Section 6.5?

Composition 
accepted

Yes

No

Composition 
rejected

 
 
 
 
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