reach and developments in alternatives to animal testing · reach and developments in alternatives...
TRANSCRIPT
REACH and
developments in
alternatives to animal
testing
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Today’s presentation
• Animal testing for REACH- how are we doing?
• EU/OECD developments in alternatives relevant
for 2018 (Annex VII and VIII)
• Does industry need more guidance?
How are we doing?
- animal numbers
Substances by registration deadline
(cumulative) 2010 2013 2018 Animals used
Commission estimate (2003)
2,704 5,165 29,324 4 million (van der Jagt, 2002)
8 -13 million (ECVAM best –worst case, 2006)
How are we doing?
- animal numbers
Substances by registration deadline
(cumulative) 2010 2013 2018 Animals used
Commission estimate (2003)
2,704 5,165 29,324 4 million (van der Jagt, 2002)
8 -13 million (ECVAM best –worst case, 2006)
Actual 4, 599 8, 729
49,851 If we get 70% as many registrations in 2018 we could be looking at least 6 million animals
+70% +70%
How are we doing?
- avoidance of unnecessary testing
ECHA Alternatives reports 2011 report 2014 report
Number substances 4,599 4,130
New animal studies 1,849 3,038
Animal tests proposed 711 701
Tests -should have been proposed 107 186
Skin irritation tests -in rabbits 135 156
Eye irritation -in rabbits 188 297
Acute dermal toxicity –in rats 161 307
Skin irritation –in vitro 301 381
Eye irritation – in vitro 206 157
EU/OECD developments in alternatives
relevant for 2018
1. Skin irritation – in vitro
2. Eye irritation – in vitro
3. Acute toxicity
– Waiving - dermal
– In vitro - oral
4. Skin sensitisation – in vitro
5. Acute fish toxicity – in vitro
Can all be used under Annex IX
-Weight of Evidence
Skin irritation-
Available test methods • Corrosion (UNGHS cat 1 or NC):
– RhE (OECD TG 431, updated 2014, further revision expected mid 2015)
– TER (OECD TG 430, updated 2013, further revision expected mid 2015)
– Corrositex (OECD TG 435, created 2006, revision expected mid 2015)
Improvement in ability to sub categorise Cat 1
Increase in available models: EpiSkin™ Standard Model (SM) and EpiDerm™ Skin Corrosivity Test, SkinEthic™ RHE1 and epiCS®
• Irritation (UNGHS cat 2 or NC): – RhE (OECD 439, updated 2013, further revision expected mid 2015)
Increase in available models: EpiskinTM, EpidermTM, Skin EthicTM, LabCYTE EPI model, epiCS®
Method for dealing with MTT reducers and coloured substances
Skin irritation
-performance of the alternatives
Contact Dermatitis 2010: 62: 109–116
76% (EpiDerm) and 70% (EPISKIN)
concordance with human patch test
Out of 16 irritants in the rabbit,
only 5 were irritating to human
skin
iivs
56% concordance with human patch test
Skin irritation-Guidance/acceptance
• Appropriate testing strategy outlined in ECHA Evaluation
Report 2010 (Feb 2011) !!!
• ECHA OECD guidelines webpage :
http://echa.europa.eu/documents/10162/21650280/oecd_test_guidel
ines_skin_irritation_en.pdf (19/03/2014)
• OECD Guidance Document – IATA . No. 203 (July 2014).
• ECHA R7 guidance update -expected July 2015
• Deletion of ‘in vivo’ requirement in Annex VIII agreed in principle by
CARACAL (Jan 2015)- revised REACH text expected to be
published early 2016- in the meantime registrants must still use
Annex XI adaptation to waive study (weight of evidence)
Eye irritation-
Available test methods • Serious eye damage (Cat 1 and NC):
– ICE (OECD TG 438, updated 2013)
– BCOP (OECD TG 437, updated 2013)
– STE (OECD TG 490- expected mid 2015)
– Fluorescein Leakage Method (OECD TG 460, created 2012, Cat 1 only)
Can be used for Cat 1 (top-down strategy) or NC (bottom-up strategy)
• Not irritating (NC):
– HCE (OECD TG 491- expected mid 2015)
Available as EpiOcularTM
Can be used for NC (bottom-up strategy)
iivs
Eye irritation
-performance of the alternatives • In-house BASF study of EpiOcular™ -85% accurate with 31
literature substances and 86% accurate with 29 propriety substances. Altern Lab Anim. 39 (2011) 365 – 87.
– “The results obtained in this study, based on 60 test substances, indicate that the EpiOcular-EIT and the BCOP assay can be combined in a testing strategy to identify strong/severe eye irritants (Category 1), moderate and mild eye irritants (Category 2), and non-irritants (No Category) in routine testing. In particular, when the bottom-up strategy with the 60% viability cut-off was employed, none of the severely irritating substances (Category 1) were under-predicted to be non-irritant. Sensitivity for Category 1/2 substances was 100% for literature reference substances and 89% for BASF SE proprietary substances.”
• SkinEthic™ HCE model (OECD TG coming) - 435 consumer product substances, overall accuracy value > 82% (irritants/non-irritants). Toxicology in Vitro 24 (2010) 523–537.
Eye irritation-
Guidance/acceptance • ECHA OECD guidelines webpage :
http://echa.europa.eu/documents/10162/21650280/oecd_test_guidel
ines_eye_irritation_en.pdf (19/03/2014)
• ECHA R7 guidance - expected July 2015
• OECD guidance IATA document in preparation - expected 2017.
• Deletion of ‘in vivo’ requirement in Annex VIII agreed in principle by
CARACAL (Jan 2015)- revised REACH text expected to be
published early 2016- in the meantime registrants must still use
Annex XI adaptation to waive study (weight of evidence)
Acute toxicity-oral
• In vitro assay (NRU3T3) can be used to demonstrate lack
of oral toxicity (Annex VII):
• NRU3T3 protocol is given by ECVAM as DB-ALM Protocol
n°139. http://ecvamdbalm.jrc.ec.europa.eu
• ECVAM recommends its use in a weight of evidence
assessment to demonstrate non-toxicity:
• https://eurl-ecvam.jrc.ec.europa.eu/eurl-ecvam-
recommendations/files-
3t3/ReqNo_JRC79556_lbna25946enn.pdf (April 2013)
• NICEATM/ECVAM validation of the NRU3T3 has shown it
predicts non-toxic substances with 95% sensitivity
Acute toxicity-dermal
• The dermal toxicity test can be waived
if the substance is not classified by the
oral route (Annex VIII)
Oral route more sensitive
Out of 2,350 substances only 6 were more toxic via the dermal route
Critical Reviews in Toxicology 40 (2010): 50–83;
Regulatory Toxicology and Pharmacology 66 (2010): 30–37.
Acute toxicity-dermal
• The dermal toxicity test can be waived if the substance is not classified by the oral route (Annex VIII)
• Removal of the requirement for the dermal route for non-toxic (LD50>2,000 mg/kg bw/d) substances was agreed in principle by CARACAL (July 2014)- revised REACH text expected to be published early 2016 - in the meantime registrants must still use Annex XI adaptation to waive study (weight of evidence)
• ECHA R7 guidance already permits waiving
Skin sensitisation-
Available test methods
• Sensitiser (Cat 1 and NC) – Direct Peptide Reactivity Assay (DPRA) (OECD TG 442c,
created 2015)
– ARE-Nrf2 Luciferase Test Method (KeratinosensTM) (OECD TG
442d, created 2015)
– h-Clat (draft OECD guideline, expected 2016)
– QSARs are also well developed, for lists of models see
http://www.antares-life.eu/
Can be used in isolation to predict Cat 1 or in combination to
predict NC
Skin sensitisation
-performance of the alternatives
Several organisations have assessed the use of the in vitro
tests in combination with accuracies around 90% (J. Appl. Toxicol. 2013; 33: 1337–1352, Toxicology and Pharmacology 69 (2014) 371–
379, Regul Toxicol Pharmacol. 71 (2015):337-51.)
LLNA/GPMT is 72% predictive of human
reactions (European Commission 2000).
Skin sensitisation
-performance of the alternatives
E.g. BASF study showed that a combination of
two out of three tests gave a reported accuracy
of 94% compared to human data. Regul. Toxicol. Pharmacol. 63, (2012), 489 – 504.
BASF
Skin sensitisation-
Guidance/acceptance • ECHA OECD guidelines webpage:
http://echa.europa.eu/documents/10162/21650280/oecd_test
_guidelines_skin_sensitisation_en.pdf (24/04/2015)
• Draft OECD guidance document in progress - expected end
2015
• ECHA R7 guidance update in progress – expected early 2016
• An option to use the in vitro tests in place of the LLNA has
been agreed in principle by CARACAL (Jan 2015)-revised
REACH text expected to be published early 2016 -In the
meantime registrants must use Annex XI adaptation to waive
study (weight of evidence).
Acute fish toxicity-
Available test methods
• Acute toxicity (LC50)
– Fish Embryo Acute Toxicity (FET) Test (OECD TG
236, created 2013)
– Fish short-term toxicity test on embryo and sac-fry
stages (OECD 212, created 1998) -not considered
live animal
– Good QSAR models for acute fish toxicity, see
http://www.antares-life.eu/
From ECVAM website
Acute fish toxicity
-performance of the alternatives
• Validation of the FET in hundreds of chemicals
demonstrated extremely strong (r=0.95)
correlation between embryo toxicity and adult
fish toxicity LC50 values. Environ Toxicol Chem. 32 (2013) :1768-83.
Taken from ECVAM validation report 2012
Acute fish toxicity-
Guidance/acceptance • ECVAM recommendation supported its use to replace acute
fish toxicity (2014) http://publications.jrc.ec.europa.eu/repository/bitstream/111111111/3
2164/1/eur%2026710_eurl%20ecvam%20zfet%20recommendation
__online.pdf
• ECHA R7 guidance on acute aquatic toxicity includes a
placeholder: – “The ZFET is specifically mentioned as a possible alternative provided
that it is fully validated and available as a standardised method (e.g.
OECD test guideline), a requirement now met by the availability of
OECD TG236 Fish embryo acute toxicity (FET) test.” (ECVAM
recommendation text 2014)
• No test method for 9.1.3 (acute fish toxicity in Annex VIII) is
specified
Does industry need more
guidance?
• More companies, more substances
• Smaller, less experienced companies?
• Substances with less data?
• Animal tests were not always avoided for
2010/2013
• Newer methods have come on board since
REACH was drafted/operational, e.g. since 2008