buav sciencereport screen

7/28/2019 Buav Sciencereport Screen

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Meeting the Deadline othe 2013 EU Marketing BanA Scientifc Review o Non-

Animal Tests or Cosmetics


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Introduction

In 2003, the European Parliament voted to ban testing on animals

or cosmetic purposes. The seventh amendment to the Cosmetics

Directive (Directive 76/768/EEC) (now recast as Regulation 1223/2009)banned testing on animals in the EU rom March 2009 and also banned

the marketing (i.e. import and sale) o products and ingredients tested

on animals outside Europe ater that date.

A postponement o the marketing ban was provided or three animal

tests (endpoints) - toxicokinetics, repeated dose and reproductive

toxicity until 2013, as it was considered at the time that these tests

were harder to replace. In addition, as a last minute compromise by the

European Parliament and Council o Ministers, the Directive provides

(in Article 4a, 2.3) that i alternatives or those three endpoints are not

available by 2013, the European Commission would put orward alegislative proposal. One o the options open to the Commission will be

to extend the 2013 deadline.

The Commission has started the process o reviewing the status o

non-animal alternative methods to these three tests. In the summer

o 2010, they asked a group o experts to report on the availability o

alternative methods or cosmetic testing. Unortunately, their resulting

drat report is both incomplete and overly negative. Similarly, the

SCCS (the Commissions Scientic Committee on Consumer Saety)

is also overly conservative in its ailure to accept some methods that

have already been shown to be scientically sound, even by ECVAM

(the European Centre or the Validation o Alternative Methods), the

European Unions body that validates alternatives.

This report provides the BUAVs analysis o the status o alternative

methods and thereore, we believe, sets out the genuine likely impact o

the 2013 marketing ban on the cosmetic industry. We conclude that the

scientic case or an extension to the 2013 deadline is not made out.

Indeed, extending the deadline would undermine the excellent work

done by industry to meet the deadline. Resources should instead be

provided to complete the validation and acceptance o the remaining

non-animal alternatives in time or 2013.

Most people [in industry] do

not even know thatin vitro

methods exist. Maybe theyhave heard something about

some movements that want to

eliminate laboratory animals,

but they relegate them to the

sphere o non-global stu

and anaticism. They cannot

imagine thatin vitro methods

may have better scientifc

validity thanin vivo tests.

Dr Costanza Rovida, Centre orAlternatives to Animal Testing

Europe, University o Konstanz,

Germany1

Who we are

The British Union or the Abolition o Vivisection (BUAV) is an international animal protection

organisation working with citizens, regulators and elected politicians to end animal

testing. We lead the European Coalition to End Animal Experiments (ECEAE), Europes

leading alliance o animal protection groups peaceully campaigning on behal o animals

in laboratories. The ECEAE draws together 17 organisations with a range o legislative,

scientic and political expertise and has been the driving orce behind campaigning

eorts to end the testing o cosmetics on animals in the EU. We are registered stakeholder

observers at the Member State Committee and the Risk Assessment Committee at the

European Chemicals Agency. With our international partners, we are also registered expertsat the OECD (the Organisation or Economic Co-Operation and Development), as the

International Council or Animal Protection at the OECD.

ECEAE

www.eceae.org

www.buav.org


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Availability and use o non-animalalternatives: the impact o the2013 deadline

1. The existence o non-animal alternatives

Table 1 lists the status o the opportunities to avoid or replace animal testing or all the toxicological endpoints (i.e. tests) which

are relevant to cosmetics testing and which traditionally use animals. More detailed inormation on alternative approaches or theendpoints subject to the 2013 deadline is provided in the subsequent sections o this report. The Commission actually considers

that two other endpoints (carcinogenicity and skin sensitisation) are also subject to the 2013 deadline. Whilst we believe this

approach is incorrect, we have also provided more detailed inormation on these two endpoints in addition to repeated dose,

toxicokinetics and reproductive toxicity.

Many o the animal tests historically used to test cosmetic ingredients have now been replaced. Some non-animal tests havebeen approved or use by the regulatory authorities; some have yet to be ormally approved. The correct legal test as to whether

there should be an extension to the 2013 deadline is whether non-animal methods should, based on scientic evaluation, have

regulatory approval or use in cosmetics, or more accurately (given sucient political will) whether they should have it by 2013

not whether they actually have it at present, some years beore this date.

It is not, thereore, sucient merely to pronounce that alternatives are not available, based on current regulatory status. Regulatoryacceptance is o course part o the picture, but the act that an alternative has not yet been accepted by regulators does not mean

that (a) it does not yet exist or (b) it is not scientically valid and suitable or testing cosmetics. Regulatory approval should ollow

scientic validation -but regrettably, very oten does not, at least not without considerable delay.

It is also important to remember that the animal tests the alternative methods replace are themselves invariably inadequate. Thetask is not to replace a perect model. As evidence or this increasingly accepted view, this report gives examples o the poor

predictivity o the existing model set against the evidence or the strength o the new non-animal alternatives. It is undamental to

this whole exercise that more cannot be expected o non-animal methods, in terms o predicting saety problems, than the existing

animal methods are able to deliver. Any other approach is legally fawed.

2. Requirements o the Cosmetics Directive

Under the Cosmetics Directive, cosmetic products and their ingredients have to be sae or consumers to use. It is the responsibilityo cosmetic companies to make sure they have the saety data to support the use o their products and ingredients. Annex I o

1223/2009 outlines the cosmetic product saety report and the tests that have to be included in this. While this Regulation does

not replace the Cosmetics Directive until 11 July 2013, it does represent current practice. It states that data has to be provided

or all relevant toxicological endpoints. Skin and eye irritation, skin sensitisation and photo-induced toxicity (in the case o UV

absorption) are specied (along with a no observed adverse eects level [NOAEL], which usually comes rom a 90-day repeated

dose study), but other endpoints are not.

Consistent with this, the Notes o Guidance rom the SCCP (the Scientic Committee on Consumer Products, now the SCCS),

state that carcinogenicity, reproductive toxicity and toxicokinetics are not considered the core data requirements. These

studies are only expected when considerable oral intake or dermal absorption is expected 2.

In a review o dossiers made by the SCCS between 2000 and 2006, less than 40% o dossiers had carcinogenicity studies and

less than 50% had toxicokinetic data. Despite this, the SCCS rarely requested to see any additional data on these or any o the

other 2013 endpoints under discussion3. The SCCS only reviews a limited proportion o cosmetic ingredients (e.g. colourants,

preservatives and UV lters) that are considered to be o greater concern. It might, thereore, be assumed that the conduct o

these tests or all other ingredients is even less likely.

The BUAV believes that the 2013 EU marketing deadline can be met, with minimal impact to

the cosmetic industry. There are three reasons or this:

1. Non-animal alternatives do exist or the remaining tests. The remaining obstacles are largely

bureaucratic and can be overcome with sucient investment.

2. The requirements o the Cosmetics Directive do not speciy animal testing or two o the three

remaining tests (toxicokinetics and reproductive toxicity).

3. The TTC approach (Threshold o Toxicological Concern) can be saely used to show that

testing is not necessary or many ingredients, due to the low exposure o consumers to individual

cosmetic ingredients.


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Alternatives: a summary othe impact o the 2013 deadline

The reduction o animal

testing is a political goal and

should not be dominated byscientists who oten cannot

accept any uncertainty, not

realising that by that behaviour,

they are only preventing

change.

Dr. Cornelis Johannes (Kees)

van Leeuwen, ormer Director

o the Institute or Health and

Consumer Protection, European

Commission, The Netherlands6

Inclusion o two additional animal tests under the 2013 deadline

Despite objections rom animal welare groups, the Commission, since 2004, assumed in its reports that that the

extension or repeated dose tests also includes two urther animal tests: skin sensitisation and carcinogenicity.

Its argument appears to be that these tests can also be considered repeated-dose toxicity, because animals may besubjected to more than one dose o the substance in question.

The BUAVs very strong legal advice is that this is unsustainable. Carcinogenicity and skin sensitisation are always

listed in EU legislation as discrete endpoints (in the text o REACH, the Test Methods Regulation, the Pesticides

Directive, the Biocides Directive, the Medicines Directive and the Veterinary Medicinal Products Directive, or

example). The Commissions own Scientic Committee on Consumer Saety (SCCS), in its notes on guidance or the

testing o cosmetic ingredients and their saety evaluation, also considers these endpoints separately.

There is no written evidence rom the time to suggest that the European Parliament and Council o Ministers intended

that the term repeated dose be used to cover several animal tests as the Commission claim. The BUAV has

questioned the Commission on this and intends to challenge any proposal to extend the deadline or these additional

endpoints. The eect o including animal tests not previously included is to reopen the debate already concluded by

the European institutions and to subvert the previous decision. In our view it is clear that the discussion should belimited in scope to the tests specied by the decision.

3.The TTC approach (Threshold o Toxicological Concern)

Should there remain a number o relatively rare cases where the remaining animal testsare still considered relevant; the TTC approach proposed by COLIPA4 (the European

Cosmetics Association) will urther reduce the number o ingredients aected. The TTC

approach is based on the concept that or all substances, there is a level o exposure

below which there is hardly any risk to human health, regardless o how toxic the

substance is. The level o exposure depends on very broad classes o likely toxicity- those chemicals not at all likely to be toxic can have higher exposure. The nature o

cosmetic ingredients means that many, such as preservatives, ragrances and dyes,

are present in only tiny amounts within a product. It is possible, thereore, to determine,

or many ingredients, that exposure will never exceed the TTC (even repeated daily

exposure to a cream, or example). What is required, in place o new animal tests, is

an evaluation (based on chemical structural similarity to other substances) as to thelikely risk, ollowed by a calculation o maximum daily exposure.

The TTC concept was rst used or ood additives, but, research by COLIPA hasshown it to be relevant or cosmetics5 and examples are now available. The SCCS is

currently reviewing its useulness. As the calculations are necessarily conservative,

this concept could mitigate the perceived need or animal tests or a great manycosmetic ingredients and yet provide the required protection to consumers.

Recommendations

As this report demonstrates, there are insucient scientic grounds or any extension to the 2013 deadline. Extending the

deadline will not signicantly help the cosmetic industry. Any extension would instead remove the incentive to validate and

accept the alternatives that already exist. This would be a highly retrograde step.

The BUAV is thereore calling upon:

The European Commission, members o the European Parliament and the Council o Ministers to hold

frm to the 2013 deadline.

The European Commission to speed up the regulatory validation and acceptance o those non-animal

alternatives that already exist.


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European CommissionExperts Report on the Statuso Alternative MethodsIn May 2010, the Commission brought together a small selection o experts toproduce a report into the status o alternatives or the 2013 deadline. By July 2010,

the Commission had published a drat version o this report, which was then open

or public consultation. The BUAV submitted signicant technical criticisms o the

contents o the report, as did a number o leading experts in non-animal methods whohad not been invited to contribute.

We have a number o concerns about the content o the drat chapters that were

produced. In summary, these were:

Incorrect approach

It is a mistaken but, unortunately, commonly held assumption that alternative

methods must seek to replicate the entire whole body response in order to replaceanimals. This stems rom the belie that, aside rom humans themselves, animals areautomatically superior models o the human response. Not only does this ignore the

act that the whole body response is currently being tested in the wrong animal,

but it ails to recognise that not all aspects o the mechanism o toxicity need to be

covered by a model in order or it to be highly predictive and, critically, useul or

regulatory purposes.

Alternatives that mimic only one aspect o the traditional animal test can be extremely

predictive, because they mimic thekey parto the mechanism. Examples o such highly

predictive tests that were overlooked in the experts report include the embryonic stem

cell test or (developmental) reproductive toxicity, the peptide reactivity tests or skin

sensitisation and the cell transormation assays or carcinogenicity. The appropriateapproach is whether alternative methods are predictive o human responses to

the same (or better) extent than animal models, rather than how complete theyare considered to be.

Incomplete aspects to the report

Disappointingly, the experts report also ailed to cover all relevant aspects or the

animal tests covered. Key alternative approaches, such as the TTC concept or theuse o QSARs, were poorly covered or even omitted rom some chapters o the repor t,

perhaps refecting the limited expert base used.

The experts also ailed to reproduce the data on the validity and applicability

o the alternatives discussed. This inormation is vital or a proper evaluation o

whether these methods are suitable, particularly in cases where they have not yet

received regulatory approval. In some cases the experts dismissed some well-established alternative methods, such as the embryonic stem cell test or

reproductive toxicity. I data on validity and applicability had been presented,

the reasons or the experts dismissal o some methods may have been

clearer.

We hope and anticipate that the fnal experts reports will take

greater account o the positive prospects or non-animal

methods.

Our report seeks to provide a more complete, qualitative

assessment o the availability and suitability o non-animal

alternatives or cosmetics testing.

It is hoped that in doing so it will redress the balance and deal with

some o the gaps in the expert report upon which the European

Commission has so ar relied.

The perceived ultimate

challenge [o complete

replication] is almost a

guarantee o not being able to

replace animal testing by 2013;

I see the problem dierently.

Dr Dave Roberts, skin

sensitisation specialist, Liverpool

John Moores University, UK8

Without substantial revision,these fve drat chapters

cannot provide a credible

basis or the Commissions

report to the European

Parliament and the European

Council.

Proessor Michael Balls, ormer

head o ECVAM and Dr Richard

Clothier, alternatives expert at

FRAME7


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Status o replacing animals orcosmetics testingThis table lists the status o the opportunities to avoid or replace animal testing or all the toxicological endpoints (i.e. tests) which

are relevant to cosmetic testing and which traditionally have used animals. Green cells indicate no impact o the 2013 deadline

due to: (a) scientic evidence that the non-animal test in question is valid and reliable (pre-validated), which has been reviewedby an authority (validated) and/or regulatory guidelines have been approved (listed as internationally accepted OECD TGs - TestGuidelines); (b) test is not a specied requirement under the Cosmetics Directive; or (c) ability to use the TTC approach.

Endpoint What alternative methods are

available?

Is testing specifed by the

Cosmetics Directive?

Can the TTC

approach be used?

Testing and marketing banned rom 2009

Dermal absorption in vitro skin test (OECD TG 428) Specied n/a

Skin irritation Reconstituted human epidermal

skin models (OECD TG 439)

Specied n/a

Eye irritation BCOP/ICE ex vivo eye models

(OECD TG 437/438 partial

replacement)

Reconstituted human corneal

eye irritation models (pre-

validated)

Top down-bottom up approach

(pre-validated)

Specied n/a

Phototoxicity 3T3 NRU cell-based test

(OECD TG 432)

Specied or some

situations

n/a

Acute toxicity (oral, dermal,

inhalation)

Battery o cell tests

Derivation rom repeated dose

inormation

Not specied, rarely

conducted because

repeated dose covers this

n/a

Mutagenicity/ Genotoxicity Battery oin vitro cell tests

(OECD 471/476/487/473)

Not specied YES

Skin sensitisation* Peptide reactivity DPRA test

(pre-validated)

Skin immune cells MUSST/

hCLAT (pre-validated)

QSAR models (validated)

Specied YES

Carcinogenicity* Battery o in vitro

cell tests (OECD

471/476/487/473)-genotoxic

carcinogens

Cell transormation assay (pre-

validated) - all carcinogens

Not specied, rarely

conducted because

repeated dose/mutagenicity

covers this

YES

Testing banned rom 2009; marketing banned rom 2013

Toxicokinetics* PBTK computer models

Battery o cell tests including

skin absorption and liver cell

metabolism (OECD TG 428/417

allows use)

Not specied YES

Repeated dose (28 or 90

day)*

Battery o cell tests Specied YES

Reproductive toxicity* Battery o cell tests (ReProTectstudy, includes validated and

pre-validated assays)

Not specied YES

TABLE 1: Status o the opportunities to avoid or replace animal testing

*These endpoints are covered in more detail in subsequent sections


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Skin sensitisation

As previously outlined, the BUAV believes that the Commission has no power to

propose postponement o the marketing ban in relation to this endpoint as the ban

correctly came into orce in 2009. The ollowing inormation demonstrates that there

would be, in any event, no scientifc justifcation or a postponement.

Endpoint: Skin sensitisation is an allergic reaction to a particular substance that

results in the development o skin infammation and itchiness. The skin becomes

increasingly reactive to the substance each time it is exposed to it. The animal tests

involving mice or guinea pigs only predict human reactions 72% o the time9.

Alternatives: The mechanism o how skin reacts to sensitising substances is actuallywell understood. The key step is the reaction o proteins in the skin to the substance, a

process called haptenation. It is thereore possible to determine the skin sensitisation

potency o a substance based on how it binds to proteinsin vitro (i.e. in a test tube).

One o these protein reactivity tests (the Direct Peptide Reactivity Assay DPRA) has

been used by industry since the early 2000s and has almost completed ECVAM pre-

validation. There is already evidence that this test alone can correctly predict 89%o substances. QSAR (Quantitative Structure-Activity Relationship) computer models

alone also have similar predictive strength. In addition, twoin vitro methods using skin

cells (MUSST and hCLAT) are also being pre-validated by ECVAM, with results due

in 2011.

Analysis o the situation: The peptide reactivity test is already in use in cosmetic

companies10 or prediction, risk assessment and classication purposes. Experts

believe that this test alone will suce; developing models that consider metabolism

would only underestimate the risk to humans11. Indeed, researchers rom COLIPA

recently stated: The replacement o the need or animal testing or skin sensitisation

risk assessment is viewed as ultimately achievable and the next couple o years shouldset the timeline or this milestone12.

Whether a chemical is

a sensitiser or not, and

how potent it is i it is asensitiser, depends on

its chemical properties

and on nothing else.

Dr Dave Roberts, skin

sensitisation specialist,

Liverpool John Moores

University, UK13

Alternative Evidence o validity Status

Direct Peptide

Reactivity Assay

(DPRA)

94% agreement within vivo data on 18 chemicals14

89% agreement within vivo data in 82 chemicals15ECVAM pre-validation ongoing (results

expected 2011)

Improved sensitivity with weak

sensitisers16

MUSST (in vitro cell

activation)

93% o 16 chemicals correctly predicted in Procter &

Gamble (P&G) study17

Used by LOral on more than 800 chemicals:

acceptable or 80% o them18

ECVAM pre-validation ongoing (results

expected 2011)

Improvements or water-insoluble,

coloured, toxic substances, metabolism

have been made

hCLAT

(DC activation)

Evaluated by ve labs (P&G, Shiseido, Kao, Henkel

and LOral) since 200419

Studies at Shiseido show 93% correct predictions in

29 chemicals20 and 84% agreement in 100 chemicals21

JaCVAM (lead) - ECVAM pre-validation

study ongoing (results expected 2011)

QSAR computer

models

83% correct classications or DEREK; 73% or

TOPKAT22

TOPS-MODE used to screen 229 hair dyes23

CAESAR made 90% correct predictions on 42

chemicals24

OECD Toolbox has data on 600-800 substances or

read across

Accepted or regulatory purposes

or REACH as long as each model is

validated according to OECD principles

TABLE 2: Alternatives or skin sensitisation


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Carcinogenicity

As previously outlined, the BUAV believes that the Commission has no power to

propose postponement o the marketing ban in relation to this endpoint as the ban

correctly came into orce in 2009. The ollowing inormation demonstrates that there

would be, in any event, no scientifc justifcation or a postponement.

Endpoint: A carcinogen is a substance that causes cancer or increases the likelihood

that someone will develop cancer. The animal test is a long (two-year) and unreliable

study with an estimated predictivity o only 42%25.

Alternatives: Cancer is oten caused by substances that damage the genes in the

cells o the body (so-called genotoxic carcinogens). These types o substancescan be identied by a number o long-standing in vitro cell-based tests. These tests

have been alleged to be over-sensitive, but more recent tests are more predictive. A

more complete method based on Cell Transormation Assays (CTA) using rodent cells

(Syrian Hamster Embryo (SHE), Balb/c3T3 and Bhas42 cells) has also been in use

or over 40 years, but has only just entered an ECVAM pre-validation study. These

assays can detect all known types o carcinogens and have shown to be up to 95%predictive.

Analysis o the situation: Experts agree that carcinogenicity studies are rarely

conducted, as they are expensive and time-consuming26. In addition, carcinogenicity

tests are not specied by the Cosmetics Directive and are rarely requested by theSCCS27. A combination o the acceptedin vitro genotoxicity tests, the CTA assay and

exposure-based TTC approaches (providing a precautionary approach or consumers)

should be the preerred approach.


Genotoxic carcinogens

Bacterial Reverse Mutation

Assay (Ames test)

Developed in the late 1950s. Well established

and scientically accepted test

90% o rodent carcinogens detected when

combined with MLA and MNT assays29

77% accuracy on 368 chemicals30


(OECD TG 471, 1997)

In vitro gene mutation assay in

mammalian cells (MLA)

90% o 553 rodent carcinogens detected when

combined with MNT and Ames test31


(OECD TG 476, 1997)

In vitro chromosome aberration

assay in mammalian cells (CA)

85% o 553 rodent carcinogens detected when

combined with Ames test and MLA32Accepted or regulatory purposes

(OECD TG 473, 1997)

In vitro micronucleus assay inmammalian cells (MNT)

90% o 553 rodent carcinogens detected whencombined with MLA and Ames test33

83% agreement on 113 chemicals in ECVAM

validation study34

Validated by ECVAM 200635


(OECD TG 487, 2010)

Genotoxic and non-genotoxic carcinogens

Cell transormation assays

(CTA with SHE, Balb/3T3 and

Bhas42 cells)

Assays established since late 1960s

OECD review in 2007 concluded that 90-95%

human carcinogens could be detected36

ECVAM workshop ound that 80-83% rodent

carcinogens were detected on 213 chemicals37

P&G study showed 85%agreement with rodent

data with 56 chemicals38

Pzer study showed 89% agreement with rodentdata with 19 chemicals39

Development o test guideline

recommended by OECD in 200640

ECVAM pre-validation completed

in 2009 or SHE and Balb/3T3,

ongoing or Bhas42 (statement

was expected in 2010)

The two-year cancer bioassay

is rarely perormed due to its

high cost and poor reliabilityor cosmetic ingredients,

besides animal welare

reasons. A combination o

tests, includingin vitro cell

transormation assay(s) and,

eventually, toxicogenomic

approaches, could be a more

cost-eective tool or cosmetic

ingredients.

Dr Annamaria Colacci, Centre orEnvironmental Carcinogenesis

and Risk Assessment,

Environmental Protection and

Health Prevention Agency, Emilia

Romagna Region, Italy28

TABLE 3: Alternatives or carcinogenicity


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Toxicokinetics

Endpoint: Toxicokinetic studies are conducted to obtain inormation on how a substance is absorbed, distributed, metabolised

and excreted by the body (also known as ADME studies). This is generally considered to be nice to know inormation as it helps

to identiy the likelihood o adverse eects in the body. Absorption is whether the substance crosses the bodys natural barriers toget inside the body (i.e. through the skin, lungs or gut); distribution is how the substance is distributed inside the body (i.e. within

blood and tissues); metabolism is whether the liver breaks down the substance into dierent substances; and excretion is how the

substance is eliminated by the body, mainly via the kidneys and then the urine. Animals have signicantly dierent metabolism and

physiology to humans. As a result, beorein vitro ADME studies on human cell models were routinely used by the pharmaceutical

industry, the ailure rate o drugs in clinical trials due to poor prediction o ADME was 40%41 - now it is only 10%42.

Alternatives: Relevant stages o toxicokinetics can be modelled using mathematical physiologically-based toxicokinetic models

(PBTK). These models consist o a set o physiological and chemical parameters that can predict the distribution and excretion

o substances through the human body ollowing initial input o inormation on absorption and metabolism. They have been

used by the pharmaceutical industry with growing sophistication since the 1970s43. The skin is the main route or the absorption

o cosmetics and can already be modelled using the regulatory approved in vitro skin method. Metabolism can be predictedthrough the use o high-throughput assays on cultured human hepatocytes (liver cells). These assays are commonly used in most

pharmaceutical companies.

Analysis o the situation: Toxicokinetic studies are not a legal requirement or the saety assessment o cosmetics under the

Cosmetics Directive and appeared in less than 50% o dossiers recently presented to the SCCS44. The use o PBTK computer

models, coupled within vitro dermal absorption and metabolism data, can adequately replace the key components. Indeed, theoption to use PBTK models and in vitro assays on liver cells to address metabolism has been included in the recently updated

OECD TG 417 on toxicokinetics.


Absorption

In vitro dermal absorption test In vitro-in vivo correlation evidenced since early

1980s45

OECD experts agreed in 1999 that there was

sucient data to support the Test Guidelines46

Validation study on new reconstituted human

epithelial models demonstrated appropriateness

on eight OECD test chemicals47

Basic criteria or the use or cosmetics

rst published by SCCNFP (now SCCS) in

199948

Accepted or regulatory purposes (OECD

TG 428, 2004)

Distribution and excretion

PBTK computer models 80% correctin vivo predictions o distribution or

123 drugs within two-old error49

70% o 19 human drugs would have been

predicted or pharmacokinetics by PBPK models

alone50

90% correct predictions o renal excretion or 40

compounds51

88% precision o predicted renal clearance or

141 drugs52

Use proposed by EFSA or pesticide

residues in ood53

Use included in regulatory guidelines

(OECD TG 417, 2010)ECVAM workshop in 2007 set guidelines

or their use54

Metabolism and excretion

In vitro assays on hepatocytes

(liver cells)

Review o studies concluded that hepatic

clearance could be predicted using human liver

microsomes55

Retrospective analysis on 50 drugs ound that

human liver cells are as predictive as animaltests56

In vitro tests with PBPK modelling (SCHH-PBPK)

gave better prediction accuracy or humans

compared toin vivo rat and dog tests57

Being pre-validated by ECVAM in 2011

Included in regulatory guidelines (OECD

TG 417, 2010)

TABLE 4: Alternatives or toxicokinetics


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Repeated dose

Endpoint: Repeated dose toxicity tests assess any persistent or progressive

dysunction o cells, organs or systems resulting rom long-term exposure to a

substance. The liver is the primary site o potential toxic injury (hepatotoxicity) and

species dierences in the activities o the liver are one o the major contributors to

the species dierences observed in the toxicity o chemicals and drugs. Severalreviews o the ability o rodent tests to predict human toxicity, mainly in the area o

pharmaceuticals, have ound that they are only about 40-60% predictive58, 59.

Alternatives: Several in vitro models, developed as stand-alone methods, are at

various development/validation stages in relation to most common targets or toxicity

(see Table 5). Although studies have shown that these tests can predict eects seenin human organs, the practical issue is how to combine the results rom several tests

into a single saety actor or risk assessment purposes.

Analysis o the situation: Repeated dose inormation (No Observed Adverse Eects Level, NOAEL) is required or new cosmetic

ingredients but in many cases this can be avoided by use o the TTC concept, as substances are used in such low quantities that

no adverse eects would be expected. In instances where this cannot be achieved, a battery oin vitro tests should be employed ocusing on the liver, which is the key target organ or repeated dose toxicity, ollowed by kidneys, heart, nerves, lung and immune

system and selecting the more sensitive endpoint or the determination o the NOAEL61. QSAR computer models can also be

used62, 63.


Hepatotoxicity (liver)

In vitro hepatotoxicity on human liver cell

lines

Pzer study ound 80% o 243 human

hepatotoxicants detected64

100% o ten hepatotoxicants detected

65

Requires validation studies

Long-term cell lines and cultures now

availableNephrotoxicity (kidneys)

In vitro kidney cell lines Good prediction o 15 nephrotoxicants

in vitro66

ECVAM recommended validation

studies in 199467

Ongoing internal validation at MerckSerono68

Cardiotoxicity (heart)

In vitro heart cells 81% agreement betweenin vitro

and clinical cardiotoxicity on six

compounds69

Up to 97% agreement within vivo or

our cardiotoxicants70


Neurotoxicity (nerves)In vitro neuronal cell test Excellent agreement within vivo or

organophosphorus compounds71Ring trial ongoing with EU and US labs72

Pulmonary toxicity (lungs)

In vitro lung epithelial cells

EpiAirway

MucilAir

> 81% correlation with existing human

data with 11 chemicals73 on MucilAir


Immunotoxicity

CFU-GM (rom bone marrow cells) Accurate prediction oin vivo with

ve out o six test substances in

pre-validation study. Positive resultsobtained on additional 20 substances74

Validated by ECVAM in 2000 (ESAC

statement 2006)75

In vitro human whole blood cytokine

assay

Results correlated well with thein vivo

data on 31 compounds76

ECVAM pre-validation in 2002

In vitro lymphocyte prolieration assay 100% correct predictions on six

chemicals77

Progressing towards pre-validation78

It is clear that the use o

animals has limitations; we are

not 70kg rats.

Dr Thomas Hartung, ormer Head

o the European Centre or the

Validation o Alternative Methods,

Italy60

TABLE 5: Alternatives or repeated dose


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Page 11

Reproductive toxicity

Endpoint: Reproductive toxicity reers to a wide variety o adverse eects that may

occur in dierent phases within the reproductive cycle, including eects on male and

emale ertility, sexual behaviour, embryo implantation, embryo development, birth

and growth and development o the young. Animal tests or reproductive toxicity take

a long time and use thousands o animals. In addition, a number o studies have shownthat they only detect about 60% o known human reproductive toxicants79, 80.

Alternatives: A number o methods, including whole embryo cultures, stem cell tests

and receptor binding assays, have been developed and are either validated according

to ECVAM principles and/or are already OECD guidelines. Individually, some o these

methods already show sucient predictability across a range o test chemicals thatshould be sucient or regulatory purposes. It may not be necessary to cover all

stages o the reproductive cycle as some are more sensitive to chemicals than others -

or example, the EST (embryonic stem cell test) covers the development o the embryo,

which is a very sensitive period.

Analysis o the situation: A combination o these methods, covering the mostsensitive endpoints in the reproductive cycle, could now predict reproductive toxicity

to an acceptable level o certainty. Indeed, the EU ReProTect project has recently

concluded that a battery o cell tests allowed a robust prediction o adverse eects

on ertility and embryonic development81, with a combined accuracy o between 70

and 100% or ten test chemicals82. The use o these tests should be viewed in thecontext o the poor predictivity o the animal test and the act that due in part to the

low exposure o humans to individual cosmetic ingredients these tests are not in any

case specied by the Cosmetics Directive. Those companies that voluntarily undertake

reproductive toxicity tests usually only carry out the developmental toxicity test83,

which the EST eectively replaces. In addition, the TTC approach, whose easibility

or reproduction endpoints has been demonstrated or chemicals generally84 and iscurrently under review or use on cosmetics, can also be used.

See Table 6 overlea or alternatives to reproductive toxicity.

It cannot be acceptable [to

the Health and Consumers

Directorate-General] thatthe promising results o the

ReProTect easibility study,

which was unded by the

Commissions Research DG,

are not presented [in the

experts report].

Proessor Horst Spielmann,

Proessor or Regulatory

Toxicology at the Freie Universitt

Berlin, Germany, and advisor tothe President o the Federal BR,

Germany85


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Page 12

Reproductive toxicity


Embryonic development

Ex vivo rodent whole embryo

culture test (WEC)

Micromass test (MM)

Widely used by industry or screening

developmental toxicants

ECVAM validation study: up to 80%accuracy with 14 chemicals (100% accuracy

with strong embryotoxicants)86


statement 2002)

Mouse/human embryonic stem

cell test (EST)

Widely used by industry or screening

developmental toxicants

ECVAM validation study: 78% agreement or

14 chemicals (100% or strong embryotoxic

chemicals)87

75% agreement within vivo or 63

chemicals88

88% accuracy or eight drugs89


statement 2002)

Improvements have recently been made

to increase applicability90 and speed o the

assay91 and to account or metabolism92

Male ertility

Computer-Assisted SpermAnalysis (CASA)

Test evaluated by two dierent laboratories

on more than 35 chemicals93

Pre-validated in ReProTect project

Testicular ragment culture 82% expected results on 11 chemicals94 Needs to be taken orward or pre-validation

Leydig cell test Good results on 15 chemicals95

Detected all ve endocrine disruptors96Needs to be taken orward or pre-validation

Sertoli cell test Good results in two laboratories or seven

chemicals97

Needs to be taken orward or pre-validation

Female ertility

Bovinein vitro (oocyte)maturation (bIVM)

Good correlation within vivo eects or 15chemicals98, good inter-laboratory variability

on eight chemicals99

Pre-validated in ReProTect project

Endocrine eects

Estrogen receptor alpha binding

assay

Bayer Schering study showed it reliably

ranked compounds with strong, weak and no

eect with high accuracy on 12 chemicals100

Part o OECD/ReProTect project, expected

to go to ECVAM validation

Estrogen receptor (ER)

transcriptional activation assay,

MELN

Bayer Schering pre-validation study showed

good accuracy on 16 chemicals and good

inter-laboratory variability101

ECVAM pre-validation report due 2011,

expected to go to ECVAM validation

AR CALUX reporter gene assay Inter-laboratory study on 64 chemicals

showed 74% agreement102

Pre-validation study showed excellent

agreement or 14 out o 16 chemicals103

Up to 85% agreement with rabbit test or 50

chemicals104

Pre-validated in ReProTect (AXLR8),

expected to go to ECVAM validation

Estrogen receptor transcriptional

assay, LUMICELL-ERAll 28 estrogen disruptors were detected105 ICCVAM validation report expected 2011

Stably transectedtranscriptional activation assay

(STTA) estrogen

80% accuracy on 46 chemicals106 Validated by CERI in 2006Accepted or regulatory purposes (OECD

TG 455, 2009)

H295R steroidogenesis assays

based on a human cell line

78% accuracy or testosterone eect on 18

chemicals, 88% or estradiol eect on 16

chemicals107

Overall, these results indicate that the H295R would always fag a chemicalas a potential disruptor o steroidogenic

processes or a reproductive toxicant (OECD

2009)

Validated by OECD/EPA in 2009

Drat OECD test guideline being discussed

TABLE 6: Alternatives or reproductive toxicity


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Page 13

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2 SCCP 2006. Notes o guidance or the testing o cosmeticingredients and their saety evaluation, sixth revision. SCCP/1005/06.

3 Pauwels, M. et al. 2009. Critical analysis o the SCCNFP/SCCP

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5 ibid.

6Van Leeuwen, K. 2010. Personal communication to the BUAV.

7

Balls, M. and Clothier, R. 2010. European Commission Consultationon the Drat Report on Alternative (Non-animal) Methods or

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8 Roberts, D. 2010. Personal communication to the BUAV.

9 SCCNFP 2000. Opinion on the murine local lymph node assay

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11 Roberts, D. W. and Patlewicz, G. Y. 2010. Updating the skin

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12Aeby, P. et al. 2010. Identiying and characterising chemical skin

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13 Roberts, D. 2010. Personal communication to the BUAV.

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28 Colacci, A. 2010. Personal communication to the BUAV.

29 Kirkland, D. M. et al. 2005. Evaluation o the ability o a battery othree in vitro genotoxicity tests to discriminate rodent carcinogens

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32 ibid.

33 ibid.

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35 ESAC 2006. Statement on the scientic validating o the in vitro

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82AXLR8 2010. Alternative testing strategies progress report 2010.

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93As reerence no. 82, as above.

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100 Freyberger, A. et al. 2010. Assessment o a robust model protocol

with accelerated throughput or a human recombinant ull lengthestrogen receptor-binding assay: Protocol optimization and intra-

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Reprod. Toxicol. 30, 50-59.

101 Witters, H. et al. 2010. The assessment o estrogenic or anti-

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102 Sonneveld, E. et al. 2006. Comparison o in vitro and in vivo

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103Van der Burg, B. et al. 2010. Optimization and prevalidation o the

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104 Sonneveld, E. et al. 2010. Validation o in vitro screening models or

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in vivo activity in rabbits. Toxicol. In Vitro. [Epub ahead o print]

105 Gordon, J. D. and Clark, G. C. 2005. Submission o XDSs

LUMI-CELL ER high-throughput system or screening estrogen-like chemicals or review by ICCVAM: http://iccvam.niehs.nih.gov/

methods/endocrine/endodocs/ICCVAMSubmission28Jan05.pd.

106 CERI 2006. Drat validation report o TA assay using HeLa-hER-9903 to detect estrogenic activity: http://www.oecd.org/

dataoecd/7/27/37504278.pd.

107 OECD 2009. Multi-laboratory validation report o the h295r

steroidogenesis assay to identiy modulators o testosterone and

estradiol production, No. 132 Paris, France.


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