EU REACH regulation changed the way to do chemical risk assessment. All chemicals marketed or manufactured in the EU must have its own dossier. Non standard methods including alternatives to animal testing are accepted. Half Italian, half English

1. La rivoluzione REACH
Costanza Rovida
REACH Mastery - Como
23 maggio 2014

2. La rivoluzione REACH
Costanza Rovida
Costanza.rovida@chimici.it
Center for Alternatives to Animal Testing – Europe (CAAT-Europe)
University of Konstanz
23 maggio 2014

5. Article 1
Aim and scope
1. The purpose of this Regulation is to ensure a high
level of protection of human health and the
environment, including the promotion of alternative
methods for assessment of hazards of
substances, as well as the free circulation of
substances on the internal market while enhancing
competitiveness and innovation

6. Article 1
Aim and scope
1. The purpose of this Regulation is to ensure a high
level of protection of human health and the
environment, including the promotion of alternative
methods for assessment of hazards of
substances, as well as the free circulation of
substances on the internal market while enhancing
competitiveness and innovation

7. List of required
endpoints,
according to
REACH
Regulation
Physico-chemical
information

8. List of required
endpoints,
according to REACH
Regulation
Toxicological
information

9. List of required
endpoints,
according to
REACH
Regulation:
Ecotoxicological
information

11. IUCLID
fields to be completed

12. Guidelines on Information Requirements

13. Guidelines on Information Requirements
TOTAL of 34 FILES !!! (How many thousands of pages?)

14. ECHA Practical Guides
1. How to report in vitro data
2. How to report Robust Study Summary
3. How to report Weight of Evidence
4. How to report waiving
5. How to report (Q)SARs
6. How to report read across and categories
http://echa.europa.eu/doc/press/na_10_16_practical_guides_20100409.pdf

15. Article 1
Aim and scope
1. The purpose of this Regulation is to ensure a high
level of protection of human health and the
environment, including the promotion of alternative
methods for assessment of hazards of
substances, as well as the free circulation of
substances on the internal market while enhancing
competitiveness and innovation

16. Articolo 13

17. REACH Regulation
ANNEXES VII-X
Before new tests are carried out to determine the properties listed in this
Annex, all available in vitro data, in vivo data, historical human data, data
from valid (Q)SARs and data from structurally related substances (read-across
approach) shall be assessed first
ANNEXES VII-X – Column 2
Specific rules for adaptation from column 1
ANNEX XI
General rules for adaptation of the standard testing regime set out in
annexes VII to X

18. Annex XI
TESTING DOES NOT APPEAR SCIENTIFICALLY NECESSARY
• Use of existing data
• Historical human data
PRO and CONS
Studies are often very poor described
No analytical identification of the substance
No identification of the impurities
……………..
………………

19. Annex XI -Weight of Evidence
There may be sufficient Weight of Evidence from several independent
sources of information leading to the assumption/conclusion that a
substance has or has not a particular dangerous property, while the
information from each single source alone is regarded insufficient to support
this notion.
• newly developed test methods
• not yet included in the test methods referred to in
Article 13(3)
• different studies with the same reliability, no key study,
but consistent to assess the end point

20. Annex XI - (Q)SAR
QUALITATIVE OR QUANTITATIVE
STRUCTURE-ACTIVITY RELATIONSHIP ((Q)SAR)
PRO and CONS
Applicability domain
Fit the purpose
“Poor” training data sets
Difficulties in reporting
……………..
………………

21. Annex XI - Grouping and Read Across
GROUPING OF SUBSTANCES AND READ-ACROSS APPROACH
Two approaches:
GROUPING IN CATEGORIES (HPV-SIDS approach)
chemical similarities
functional groups
physical chemical properties
common environmental fate
common metabolites
READ ACROSS FROM SIMILAR SUBSTANCES
a specific end-point can be assessed with a result on a similar
substance
different end point can be assessed from different substances

22. Annex XI
ALTERNATIVE METHODS (ECVAM activities)
http://tsar.jrc.ec.europa.eu/
Reduction of animals: Sensitisation
Acute oral toxicity
Acute aquatic toxicity
Developing screening metods that can support the reliability of a category
or a read across
Pavia – 7 maggio 2011

23. Annex XI
GROUPING OF SUBSTANCES AND READ-ACROSS APPROACH
Pavia – 7 maggio 2011
Two approaches:
GROUPING IN CATEGORIES (HPV-SIDS approach)
chemical similarities
functional groups
physical chemical properties
common environmental fate
common metabolites
READ ACROSS FROM SIMILAR SUBSTANCES
a specific end-point can be assessed with a result on a similar
substance
different end point can be assessed from different substances

24. Annex XI
SUBSTANCE-TAILORED EXPOSURE-DRIVEN TESTING
Testing in accordance with sections 8.6 and 8.7 of Annex VIII, Annex IX and
Annex X may be omitted, based on the exposure scenario(s) developed in
the Chemical Safety Report
PERFORM a Chemical Safety Assessment complete with Exposure
Scenarios even if the substance is NOT DANGEROUS
CONS: Companies are often not cooperative even if they save money
Pavia – 7 maggio 2011

25. In vitro methods
In vitro toxicity testing is the scientific analysis of the effects of toxic chemical
substances on cultured bacteria or mammalian cells. In vitro (literally 'in
glass') testing methods are employed primarily to identify potentially
hazardous chemicals and/or to confirm the lack of certain toxic properties in
the early stages of the development of potentially useful new substances
such as therapeutic drugs, agricultural chemicals and direct food additives
that may or may not taste good.
In vitro assays for xenobiotic toxicity are recently carefully considered by key
government agencies (e.g. EPA; NIEHS/NTP; FDA), mainly due to a societal
movement to reduce the use of animals in research, and a desire to better
assess human risks. There are substantial activities in using in vitro systems to
advance mechanistic understanding of toxicant activities, and the use of
human cells and tissue to define human-specific toxic effects.
www.answers.com

26. I metodi alternativi: che cosa sono
Principio delle 3R:
Reduction = Riduzione
Refinement = Miglioramento
Replacement = Sostituzione
Russell, W. M. S. and Burch, R. L. 1959. The principles of
humane experimental technique

27. Direttiva EU 63/2010
Sostituisce la vecchia Direttiva EEC 86/609
con una maggiore attenzione al benessere
animale e un esplicito riferimento alle 3R
Maggiore controllo e rigore sugli
esperimenti

28. Element Dir 86/609/EEC Dir 2010/63/EU
Ambito di applicazione Vertebrati. Vertebrati,
cefalopdi, Feti
Compliance with the Three Rs Yes Yes
Project evaluation No Yes
Project authorisation (Yes/No) Yes
Housing and care standards guidelines binding
standards
Main differences between
the current & the new Dir

29. Main differences between
the current & the new Dir
Element Dir 86/609/EEC Dir 2010/63/EU
Project follow-up (Yes) Yes
- Retrospective assessment No Yes
Non-technical summaries No Yes
Severity classification No Yes
Ispezioni periodiche Yes Obbligatorie

30. Shell Fish Biotoxins
Mouse bio-assay

32. Shell Fish Biotoxins
Metodo in vitro

33. Confronto metodo in vitro e in vivo
140
120
100
80
60
40
20
0
In vitro Assay Mouse Bioassay
17.2% 52.6% 80.0%
1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145 151 157
μg STX eq/100 g
correlative sample number

34. In vitro methods
In vitro toxicity testing is the scientific analysis of the effects of toxic chemical
substances on cultured bacteria or mammalian cells. In vitro (literally 'in
glass') testing methods are employed primarily to identify potentially
hazardous chemicals and/or to confirm the lack of certain toxic properties in
the early stages of the development of potentially useful new substances
such as therapeutic drugs, agricultural chemicals and direct food additives
that may or may not taste good.
In vitro assays for xenobiotic toxicity are recently carefully considered by key
government agencies (e.g. EPA; NIEHS/NTP; FDA), mainly due to a societal
movement to reduce the use of animals in research, and a desire to better
assess human risks. There are substantial activities in using in vitro systems to
advance mechanistic understanding of toxicant activities, and the use of
human cells and tissue to define human-specific toxic effects.
www.answers.com

35. IN VITRO METHODS
Validated methods:
Skin corrosion/irritation
Skin absorption
Eye corrosion/irritation (partially)
Screening for genotoxicity and mutagenicity (partially)
Acute Toxicity (partially)
Pre-validation methods:
Sensitisation
Carcinogenic potential
Reproductive toxicity (embryotoxicity)

36. In vitro methods: Skin sensitisation
LOCAL LYMPH NODE ASSAY
Days 1, 2 & 3
Apply Chemical
Day 6 - Inject
3 H-Thymidine
5 hours Later
Remove Lymph
Nodes
Make Cell
Suspension
3
Determine H-Thymidine
Incorporation by Liquid
Scintillation Counting

37. In vitro approach to Skin sensitisation

38. SKIN Sensitisation
DPRA (Direct Peptide Reactivity Assay):
OECD 406 (Guinea pig): 2,000€
OECD 429 (LLNA, traditional test): 3,500€
OECD 442b (LLNA, BrdU-ELISA): 2,700€

39. Strategy: sensitisation
Metal salt Metal oxide
Pavia – 7 maggio 2011
MO
Test with OECD TG 428
Skin Absorption ≥ 0.1% Skin Absorption < 0.1%
MX2
LLNA positive
(Strong)
Negative
No classification as skin sensitiser
Positive
Classified as skin sensitiser H317
Read Across

40. Strategy: sensitisation
IN VITRO STUDIES : An example on how to assess doubtful sensitization
results on a basic dye supporting also exposure pattern.
No study available on both Oxalate and Chloride forms of the dye
Demonstration that the two salts are equivalent is missing
There is concern about the skin sensitisation potential
Negative result in an old study (1984)
Positive in a even older human patch test (1966)
• A new study performed on both salts is required
• A new study to confirm the sensitisation property is required
Pavia – 7 maggio 2011

41. Strategy: sensitisation
Pavia – 7 maggio 2011
ITS proposal:
1. Skin absorption assessment: OECD TG 428
2. In vitro sensitisation: U937 method, which passed the pre-validation
and has just started the official validation at
ECVAM (European Centre for Validation of
Alternative Methods)
RATIONALE
Skin Absorption: Only adsorbed substances can cause skin sensitisation
The results can be also exploited as a comparison between
the two salts
Sensitisation: a POSITIVE results is accepted (Annex XI:positive results
from a suitable in vitro test can be accepted)
a NEGATIVE result is also accepted: (peer review literature)

42. Strategy: sensitisationoss
Pavia – 7 maggio 2011
COSTS:
OECD TG 428, in GLP:
2,700 €/per substance
4,320 € for the two salts when tested simultaneously
U937
Performed for free at the University of Milan, as the validating committee
needs new substances to test
The final result will be confidential and will not be published
(LLNA in vivo method):
7,200 €/per substance
10,800 € for both salts when tested simultaneously

43. Strategy: full dossier
In vitro studies to assess the category of nickel compounds and verify the
representative substance to take the existing data in Read Across
BIOACCESSIBILITY BASED READ ACROSS ASSESSMENT OF NICKEL
COMPOUNDS: INHALATION TOXICITY
Sample ID Code(s)2 CAS No.
Ni
Content
(%)3
Interstitial
Bioaccessibility
(% Ni/g sample)4
Pavia – 7 maggio 2011
Lysosomal
Bioaccessibility
(% Ni/g sample)4
Acute Toxicity
(inhalation LC50,
mg/L)5
Ni Sulfate Hexahydrate N58-72 10101-97-0 22 12.80 21.35 2.48
Ni Oxide Green N9/N46 (N126) 1313-99-1 77 (81) 0.10 0.82 >5.08
Ni Sub-Sulfide N129 (N18) 12035-72-2 61 (70) 3.60 26.20 1.14
Ni Hydroxy Carbonate N128 (N109) 12122-15-5 49 (49) 1.65 47.206 >2.09 (F); 0.25 (M)7
Ni Chloride Hexahydrate N98 7791-20-0 25 8.10 25.056 NC
Ni Acetate Tetrahydrate N103 6018-89-9 24 10.90 24.856 NC
Ni Sulfamate
N104 13770-89-3 18 8.60 18.306 NC
Tetrahydrate
Ni Hydroxide N106 12054-48-7 54 0.02 55.80 NC
Ni Oxide Black N105 1313-99-1 75 0.56 24.50 NC
Ni Sulfide N97 16812-54-7 59 1.08 25.95 NC

44. Strategy: full dossier
BIOACCESSIBILITY BASED READ ACROSS ASSESSMENT OF NICKEL
COMPOUNDS: ORAL SYSTEMIC TOXICITY
Sample ID Code1 CAS No.
Ni Content
(%)2
Pavia – 7 maggio 2011
Bioaccessibility (%
Ni content
released)3
In Vivo Acute
Toxicity (oral LD50,
mg/kg/bw)4
Ni Sulfate Hexahydrate N58-72 10101-97-0 23 90.55% 362
Ni Oxide Green N9/N46 1313-99-1 77 0.33% >11000
Ni Oxide Green N112 1313-99-1 81 <LOD5 >11000
Ni Sub-Sulfide N18 12035-72-2 70 22.65% >11000
Ni Chloride Hexahydrate N98 7791 -20-0 25 89.85% 500
Ni Acetate Tetrahydrate N 103 6018-89-9 24 88.50% 550
Ni Hydroxy Carbonate N109 121 22-1 5-5 49 84.30% 2000
Ni Sulfamate
N 104 13770-89-3 18 83.40% 1098
Tetrahydrate
Ni Hydroxide N106 12054-48-7 54 26.30% 5000
Ni Oxide Black N105 131 3-99-1 75 29.60% 9990
Ni Sulfide N97 1681 2-54-7 59 9.75% NC6
Ni Fluoride Tetrahydrate7 N111 13940-83-5 32 82.35% 310

45. European projects
OSIRIS
Optimised Strategies for Risk Assessment of Industrial Chemicals through
Integration of Non-Test and Test Information
http://www.osiris-reach.eu/
Pavia – 7 maggio 2011

46. Improvements
With the first dossiers many data have been generated, that can be used to
improve also the reliability of predictions models in the next years
More in vitro metods will be developed / validated
Multidisciplinary approach within the working groups
Will ECHA be ready?
Pavia – 7 maggio 2011

47. An example for a Strategy
IN VITRO STUDIES : An example on how to assess doubtful sensitization
results on a basic dye supporting also exposure pattern.
No study available on both Oxalate and Chloride forms of the dye
Demonstration that the two salts are equivalent is missing
There is concern about the skin sensitisation potential
Negative result in an old study (1984)
Positive in a even older human patch test (1966)
• A new study performed on both salts is required
• A new study to confirm the sensitisation property is required

48. ITS proposal:
1. Skin absorption assessment: OECD TG 428
2. In vitro sensitisation: THP-1 method, which passed the pre-validation
and has just started the official validation at ECVAM (European Centre
for Validation of Alternative Methods)
RATIONALE
Skin Absorption: Only adsorbed substances can cause skin sensitisation
The results can be also exploited as a comparison between
the two salts
Sensitisation: a POSITIVE results is accepted (Annex XI:positive results
from a suitable in vitro test can be accepted)
a NEGATIVE result is also accepted: (peer review literature)

49. COSTS:
OECD TG 428, in GLP:
2,700 €/per substance
4,320 € for the two salts when tested simultaneously
THP-1 Performed for free at the University of Milan, as the validating
committee needs new substances to test
(LLNA in vivo method):
7,200 €/per substance
10,800 € for both salts when tested simultaneously

50. In vitro methods: Strategy
Existing
data
Weight of
Evidence
GOOD In vitro data
SENSE!
INTELLIGENT = INTEGRATED
Exposure
assessment
Read
across
Categories
In silico
data

51. Validation modular approach
Test definition
Within-lab. variability
Transferability
Between-lab.variability
Predictive capacity
Applicability domain
Minimum performance
standards
Reprodu-cibility
Relevance
“Standardised”
“Suitable/Adequate”
“Validated”
“Equivalent”
Hartung et al. ATLA 2004, 32:467-472

53. OECD Member Countries
Countries/Economies Engaged in Working
OECD Relationships with the OECD

54. OECD Methods
428 Skin Absorption: In Vitro Method
430 In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER)
431 In Vitro Skin Corrosion: Human Skin Model Test
432 In Vitro 3T3 NRU Phototoxicity Test
435 In Vitro Membrane Barrier Test Method for Skin Corrosion
437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants
438 Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants
439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
471 Bacterial Reverse Mutation Test
472 Genetic Toxicology: Escherichia coli, Reverse Assay
473 In Vitro Mammalian Chromosome Aberration Test
474 Mammalian Erythrocyte Micronucleus Test
476 In Vitro Mammalian Cell Gene Mutation Test
477 Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophilia melanogaster
479 Genetic Toxicology: In Vitro Sister Chromatid Exchange assay in Mammalian Cells
480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro
483 Mammalian Spermatagonial Chromosome Aberration Test
487 In Vitro Mammalian Cell Micronucleus Test

55. OECD Methods
428 Skin Absorption: In Vitro Method
430 In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER)
431 In Vitro Skin Corrosion: Human Skin Model Test
432 In Vitro 3T3 NRU Phototoxicity Test
435 In Vitro Membrane Barrier Test Method for Skin Corrosion
437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants
438 Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants
439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
471 Bacterial Reverse Mutation Test
472 Genetic Toxicology: Escherichia coli, Reverse Assay
473 In Vitro Mammalian Chromosome Aberration Test
474 Mammalian Erythrocyte Micronucleus Test
476 In Vitro Mammalian Cell Gene Mutation Test
477 Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophilia melanogaster
479 Genetic Toxicology: In Vitro Sister Chromatid Exchange assay in Mammalian Cells
480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro
483 Mammalian Spermatagonial Chromosome Aberration Test
487 In Vitro Mammalian Cell Micronucleus Test

56. OECD 437 BCOP Test
H318 Causes serious eye damage

57. Un esempio di una strategia
IN VITRO STUDIES : Un esempio di come una strategia in vitro è stata
utilizzata per la valutazione di un risultato dubbio sulla sensitization e per
avere maggiori dati sull'esposizione.
Nessuno studio disponibile su entrambi i Sali in commercio del
colorante (Ossalato e cloruro)
Mancanza di una dimostrazione che i due Sali fossero equivalenti
Dubbio che potesse essere allergenico
Risultato negativo in uno studio molto vecchio (1984)
Risultato positivo in un human patch test ancora più vecchio (1966)
• Necessario uno studio da fare contemporaneamente sui due sali
• Necessario uno studio per decidere sulla skin sensitisation

58. ITS proposal:
1. Skin absorption assessment: OECD TG 428
2. In vitro sensitisation: THP-1 method, che ha già passato la fase di pre-validation
and ed è in corso di validazione ufficiale a ECVAM
(European Centre for Validation of Alternative Methods)
RATIONALE
Skin Absorption: Solo le sostanze che vengono assorbite possono provocare
allergie. Il risultato può essere utilizzato come base per il
confronto tra i due sali
Sensitisation: un risultato POSITIVO è accettato (Annex XI: positive
results from a suitable in vitro test can be accepted)
un risultato NEGATIVO è accettato lo stesso (peer review
literature)

59. Non si conoscono
Sono più difficili da applicare
Spesso sono più costosi
Non si è sicuri che vengano accettati
Si è sempre fatto in un altro modo
Altex 27, 3/10

60. … Approccio avanzato, ma ancora
con impostazione classica…

61. The reproductive cycle
Courtesy of Michael Schwarz

62. Use of bovine gametes for
reproductive toxicity testing
2 cm
bovine ovaries
In vitro maturation (IVM) of bovine oocytes
bovine oocytes
Endpoint: %
Metaphase II
Chemical
exposure (24h)
during IVM
In vitro fertilisation (IVF) of bovine oocytes
Chemical
exposure (20h)
during IVF
Endpoint:
% 2Pronuclei
IVM, 24 h

63. dall’embrione alle cellule staminali
concepimento blastocisti (5 giorni)
Coltura di cellule
embrionali staminali

64. Stem Cell Toxicity Approach
Developmental Toxicity
Acute Toxicty

65. validation
performance
predictive
confidence
in vivo relevance

66. QIVIVE
Quantitative in vitro in vivo extrapolation
Target cells: permeability, transport, metabolic competence
Extracellular space: proteins, lipids, pH, plastic, other cells
Exposure (concentration x time)

67. Modelli PBPK

68. Report del
National Research
Council of the National
Academies
Pubblicato nel 2007
(Google: "Toxicity Testing in the 21st Century")

69. A New Paradigm: Activation of Toxicity Pathways
Biologic
Inputs
Normal
Biologic
Function
Morbidity
and
Mortality
Cell
Injury
Adaptive Stress
Responses
Early Cellular
Changes
Exposure
Tissue Dose
Biologic Interaction
Perturbation
Low Dose
Higher Dose
Higher yet
(Courtesy of Mel Andersen)

70. Progetto ToxCast

71. Toxicity Pathways
A cellular response pathway that, when sufficiently
perturbed, is expected to result in an adverse health
effect.
Set Chemicals Assays Endpoints Completion Available
ToxCast Phase I 293 ~600 ~1100 2011 Now
ToxCast Phase II 767 ~600 ~1100 03/2013 10/2013
ToxCast Phase IIIa 1001 ~100 ~100 Just starting 2014
E1K (endocrine) 880 ~50 ~120 03/2013 10/2013
Tox21 8,193 ~25 ~50 Ongoing Ongoing

72. Effect of Concentration Response on
Polypharmacology
72
Pentachlorophenol

74. “Non sopravvive chi è più forte o
più intelligente, ma chi
reagisce più velocemente ai
cambiamenti”
Charles Darwin