REACH and

developments in

alternatives to animal

testing

For more information please contact:

Katy.taylor@crueltyfreeinternational.org

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

OECD testing strategy

Taken from OECD Guidance Document No. 203

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

Its up to you!

Thank you for listening!