Meeting the Global Challenge - A Guide to Assessing the Safety of Cosmetics without Using Animals

A guide to assessing the safety of
cosmetics without using animals
Meeting the
Global Challenge

Contents | A guide to assessing the safety of cosmetics without using animals
“The leading
global
organization
dedicated to
ending the use
of animals to test
cosmetics and
other consumer
products.”

02 Introduction
03 Cruelty Free International
03 The Leaping Bunny
04 Why alternative tests?
04 What is an alternative test?
04 What are the benefits of using alternatives tests?
05 What about other regulations?
06 Product testing
07 Ingredient testing
08 Detailed analysis of each test and the alternatives
10 Standard toxicity tests
10 Skin absorption
11 Skin irritation/corrosion
12 Eye irritation/corrosion
13 Skin sensitisation
15 Mutagenicity/genotoxicity
16 Repeated dose
18 Non-standard toxicity tests
18 Phototoxicity
19 Acute toxicity
20 Carcinogenicity
21 Reproductive toxicity
22 Endocrine disruption
23 Toxicokinetics
24 Conclusion
Contents
Cruelty Free International | 01

02 | A guide to assessing the safety of cosmetics without using animals
Animal testing of cosmetics is deeply unpopular and is now banned
in many countries around the world 1
. Most notably, the European
Union (comprising of 28 countries) banned the testing of cosmetics
products on animals in 2003 and the testing of ingredients that are
used in cosmetics products in 2009. In addition, 11 March 2013 saw the
completion of a marketing ban, meaning that cosmetics products and
ingredients tested on animals outside of the EU after that date may not
now be sold there.
Largely driven by the EU’s 2013 deadline, research by the cosmetics
industry and national governments has stepped up. As a result,
there are now alternatives for the most commonly required safety
tests for cosmetics. In other cases, animal tests may not be required
depending on the type of ingredient and its intended applications.
Ethical concern has been the driver for this positive change and
governments can take comfort from the fact that the animal tests
normally used for cosmetics now have practical non-animal
alternatives. Since exports of animal-tested cosmetics to countries
with a marketing ban are impossible, it is important for countries
where a ban is not yet in place to provide a way forward for their
domestic industry.
This ground-breaking report is intended to help explain the position
for each safety test. It will help governments, politicians, regulators
and cosmetics manufacturers across the world switch to alternatives
to replace animal testing, ensuring the safest and most modern
methods are used, and that access to European and other markets
are not cut off due to dependence on obsolescent technology. Cruelty
Free International describes below the alternative approaches that are
available to replace animals and shows how they are more reliable,
faster and cheaper than the animal tests they replace.
—
1 The 28 countries of the European Union, Israel and India have all banned
animal testing for cosmetics.
Introduction
“There are now
alternatives for the
most commonly
required safety
tests for cosmetics.
In other cases,
animal tests may
not be required
depending on the
type of ingredient
and its intended
applications”

Cruelty Free International is the leading global
organization dedicated to ending the use of animals to
test cosmetics and other consumer products throughout
the world. We work with governments, regulators,
companies and partner organizations worldwide to
achieve effective long-lasting change for animals. Cruelty
Free International has placed the issue of animal testing
on the agenda of many governments for the very first time
as part of a global strategy to tackle product testing.
Products bearing the Leaping Bunny logo are certified ‘cruelty-free’
under the international Humane Cosmetics and Household Products
Standards. The Leaping Bunny is a global certification and applies
to all of the operations and sales of companies. We only certify
companies that have a policy not to test their products or ingredients
on animals for any market.
Companies are eligible for Leaping Bunny certification if they
guarantee to exclude from their formulations, cosmetics (or household)
ingredients which have been animal tested after an agreed, historical
date (a fixed cut-off date). They must also permit independent
audits to provide third-party assurance of their policy. More than
500 companies are now licensed to carry the Leaping Bunny logo,
including prominent names such as The Body Shop, Molton Brown,
Dermalogica, Paul Mitchell and Ecover.
Cruelty Free
International
The Leaping
Bunny

What is an alternative test?
Alternative methods are tests that use simple organisms
like bacteria, or tissues and cells from humans (so-called
in vitro tests), and sophisticated computer models or
chemical methods (so called in silico and in chemico tests).
Cruelty Free International considers an ‘alternative’ to be
any test method that does not use live vertebrate animals,
i.e. mammals, fish and birds. It is universally accepted that
vertebrate animals can feel pain or otherwise suffer.
Methods that use tissues from vertebrate animals who
have already been killed (often for other purposes such
as for food) are recognised internationally as alternatives
since the animal does not suffer during the test (these are
so-called ex vivo tests).
Sometimes, the term ‘alternatives’ is used for methods
which use live animals but use fewer animals or cause
less suffering. However, this is not how we believe the
general public understands the term, and it is not how we
use it in this report.
What are the benefits of using alternatives tests?
The public does not support the use of live animals.
• USA (2011): 72% of respondents agreed that testing
cosmetics on animals is unethical 2
.
• Czech Republic (2006): 72% of respondents agree
with the use of alternatives instead of animal tests for
cosmetics 3
.
• Norway (2002): 81% of respondents have a negative
opinion about cosmetics testing 4
.
• UK (1999): 88% of women want a complete ban on
animal testing for cosmetics 5
.
Being able to claim that products have been tested
without harming animals is increasingly seen across
the world as providing a positive, ethical choice to
consumers and can increase market sales.
• UK (2004): 79% of people said they would be likely
to swap to a brand that was not animal tested if they
discovered that their existing brand was tested on
animals 6
.
• USA (2011): 32% of people said they had purchased
products labeled as “not tested on animals” because
of their concern for animals 7
.
Why alternative
tests?
—
2 PCRM/ORC International 2011
3 The Public Opinion Research Centre (CVVM) for Svoboda zvírat, 2006
4 Opinion for Dyrevernalliansen, “Holdninger til bruk av dyr”, landsomfattende
omnibus av 2002
5 Opinion Research Business for BUAV and RSPCA, 1999
6 Opinion Research Business for BUAV, 2004
7 The Animal Tracker (Wave 4 – March 2011). Humane Research Council, 2011

Alternatives are usually cheaper and faster than the
animal test they replace.
The in vitro tests for skin and eye irritation can be
conducted in a day, whereas the corresponding rabbit
tests take two to three weeks. Similarly, one of the skin
sensitisation tests can be conducted in one day whereas
the corresponding mouse test takes at least six times
that. All of these tests can already be conducted at a cost
equivalent to the animal test, between 1 – 5,000 Euros.
Methods that avoid the lengthier systemic toxicity tests are
much cheaper and faster. For example, computer (QSAR)
models for bioaccumulation tests in fish can be run at
very little cost, assuming some in-house expertise, saving
an estimated 40,000 Euros. The cost of an expert to set
out a TTC approach or read across argument (explained
below) could typically be around 3,000 Euros, compared
to 300,000 Euros for a two-generation reproductive toxicity
test. The Cell Transformation Assay can cost as little as
500 Euros and can avoid the conduct of the rat cancer
bioassay which takes two years and costs approximately
one million Euros.
Alternatives are usually more reliable and accurate than
the animal tests they replace.
Modern alternative methods are required to go through
a validation process to demonstrate they are as, or
more, effective than the animal test they replace. The
performance of the alternative is compared to human
responses, where this is already known. Validated
alternative methods are published in the guidelines of
international bodies that harmonise the most common
methods to assess the safety of chemical substances,
such as the Organisation for Economic Cooperation
and Development (OECD). Alternatives will simply not
be accepted at international levels by the OECD without
sufficient evidence that they reliably detect toxic and
non-toxic substances. In contrast, it is important to note
that traditional animal tests have never been ‘validated’
for their use in reliably detecting the safety of cosmetic
ingredients. This means that there has not been an
independent, controlled assessment of whether the
animal test accurately and reliably predicts human
reactions using a set of substances for which the human
response is known. The validity of existing animal models
is assumed only, based on a long history of their use.
This is not adequate for today’s high safety standards.
What about other regulations?
It is possible that safety testing using animals is required
because of an ingredient’s other uses, for example as
a general purpose chemical or biocide. In these cases,
the alternatives described in this report can often still be
used since the toxicity tests required by these regulatory
regimes are usually the same as for cosmetics.
Legislation that bans animal testing for cosmetics
ingredients is usually sensitive to any overlap with other
regulatory requirements and should take a pragmatic
but principled view, to avoid undermining the ban and
public confidence whilst remaining workable in practice.
For example, under the Humane Cosmetics Standard
(Leaping Bunny certification), in cases where another
regulatory regime insists on the animal test we ask that
the certified company withdraws the tested substance
from its products if the predominant use of that substance
is in cosmetics. Although the testing may be to meet the
requirements of another regulation, the substance is being
marketed predominantly for the cosmetics industry, and
continued use of that substance would be misleading to
consumers who believe ‘no animal testing for cosmetics
purposes’ has taken place.

Testing the safety of finished cosmetics products has
not been carried out in the EU, and elsewhere, for many
years. Instead, cosmetics companies determine the safety
of new formulations made up of existing ingredients by
using calculations to determine overall safety factors8
.
Each cosmetics product is considered as a combination
of individual cosmetics substances. A qualified safety
assessor looks at the data on the ingredients and the
extent to which consumers are exposed to the product
and comes to a judgement about the safety of the product
as a whole.
The potential for local effects (irritation, sensitisation),
which may occur at the site of contact, needs to be
assessed alongside the potential for systemic (internal)
effects. The local effects of a product are generally more
straightforward to predict, based on existing data for
individual ingredients, experience of use, the level of
individual ingredients, the characteristics and intended
use of the product. Companies also gain additional
confidence in the local effects of their products by
performing compatibility tests using human volunteers.
Under strict ethical guidelines, and always after the initial
safety assessment, volunteers will test the products to
ensure that the product claims are justified and that there
are no skin irritation or sensitivity issues 9
. Companies may
also use the alternatives described here for skin and eye
irritation to double-check the lack of irritation potential
of the product as a whole before they conduct these
volunteer studies.
For systemic effects, Margin of Safety (MoS) values are
generated for each individual ingredient. These are
calculated based on an assessment of the exposure to
the human body of the ingredient and the extent to which
it is likely to be toxic. The ‘systemic exposure dose’ (SED) is
first calculated based on an assessment of how often and
how much of the product is used, the level of ingredient in
the product, whether the product is ‘leave on’ or ‘rinse off’
and the potential for the product to penetrate the skin. The
‘no observed adverse effect level’ (NOAEL) is then obtained
for the ingredient, which is a measure of its toxicity based
on new or existing toxicity data. The selected NOAEL is
divided by the SED to give the Margin of Safety (MoS) for
that ingredient. MoS values of 100 or greater are generally
considered to indicate an adequate level of safety;
however higher values may be required for particular
ingredients or product types.
Avoidance of contamination and impurities can be
assured by adherence to Good Manufacturing Practice
(GMP) and the relevant ISO and CEN standards for
production. Regulators can help improve cosmetics safety
by issuing lists of known dangerous substances that
should not be put into cosmetics. Regulators can ensure
there is traceability of the product and conduct market
surveillance. In vitro tests can be carried out to ensure the
product does not have a high microbial content and also
to determine if preservatives in the product will reduce
contamination.
In the event of any safety issues, companies declare the
quantities of substances in their products, demonstrate
GMP and ensure traceability. Animal tests in the scenario
of deliberate or inadvertent inclusion of ingredients not
listed on the packaging will not help identify what the
contaminants are, nor will they demonstrate why, if at all,
the animals become unwell. They therefore cannot explain
any safety issues that may have arisen as a result.
Product testing
—
8 SCCS 2012. The SCCS’s Notes of Guidance for the Testing of Cosmetic Substances
and their Safety Evaluation, 8th Revision SCCS/1501/12

9 Opinion concerning guidelines on the use of human volunteers in compatibility
testing of finished cosmetic products – adopted by the Scientific Committee on
Cosmetics and Non-food Products intended for Consumers during the plenary
session of 23 June 1999 http://ec.europa.eu/health/scientific_committees/
consumer_safety/opinions/sccnfp_opinions_97_04/sccp_out87_en.htm

—
10 http://ec.europa.eu/consumers/cosmetics/cosing/
11 Colipa response to the impact assessment of the EU marketing ban 2013.
http://ec.europa.eu/consumers/sectors/cosmetics/files/pdf/animal_testing/
at_responses/colipa_ia_2013_1_en.pdf
Since the safety of a cosmetics product relies on
information about the ingredients, information on the
safety of the ingredients is required. For products made
up of existing ingredients this is a relatively
straightforward task. For example, there are over 24,000
cosmetics ingredients listed on the EU COSING database 10
for which there are safety data available. No new animal
(or non-animal) safety data is usually required. Exceptions
to this may be ingredients which become a concern and
for which the regulators in a particular region may ask
for more data. This has been the case for some specialist
cosmetics ingredients such as hair dyes, preservatives
and UV filters.
However, the impact of ‘no animal testing’ for most
cosmetics product manufacturers is minimal. Companies
can continue to develop their products using existing
ingredients or ingredients that become available that have
been shown to be safe using non-animal approaches.
The lack of impact can be shown by the fact that the EU
ban is in place, and that over 500 product manufacturers
certified by Cruelty Free International’s Humane Standards
have already excluded from their formulations cosmetics
ingredients which have been recently animal tested.
The proportion of genuinely new ingredients entering
the market every year is actually very low. According to
Cosmetics Europe, “across the industry, new ingredients
are introduced at an annual rate of around 4% of the total
portfolio”. Only a proportion of these are thought to be
new to all uses 11
. Those companies who wish to innovate
and use genuinely new ingredients have several options:
a) to determine the safety of new but very similar
ingredients based on ‘read across’, i.e. extrapolation
of the information from data on the original
substance;
b) to use the TTC (threshold of toxicological concern)
approach to determine if any testing is really needed
due to low exposure levels;
c) to use alternative methods such as in vitro or
computer based methods like QSARs to determine
aspects of the safety of the ingredient.
Finally, companies always have the option to continue to
innovate but not use, i.e. screen out, ingredients where,
exceptionally, safety concerns cannot be alleviated based
on these approaches rather than by animal testing. In this
way human health is best protected.
Ingredient
testing

If a country or region bans animal testing for cosmetics, it can be
confident that:
• The alternatives provide as much or more safety for consumers
• The alternative tests are generally of comparable cost or cheaper
than the existing tests
• Innovation in cosmetics is not prevented.
The Table right summarises the position, and is followed by a
detailed analysis of each type of toxicity test (endpoint).
For each endpoint we provide the most acceptable, feasible and
available non-animal alternative. We have separated out endpoints
into those that are ‘standard’, i.e. commonly required by national
regulators, and ‘non-standard’ endpoints, i.e. those that are not a
routine requirement for cosmetics. These tests are often ‘triggered’
when use and exposure is particularly high or indicated for
other reasons.
Detailed analysis of each
test and the alternatives
“Alternatives provide
as much, if not
more, safety for
consumers”

Table 1:
Standard cosmetics toxicity tests and the available options to avoid animal testing
Endpoint Tests for Animal test
Options to avoid
animal test
Skin absorption
The extent to which the
substance will penetrate
the skin
The substance is rubbed onto
the shaved backs of rats and
they are killed the next day
(OECD TG 427)
Ex vivo skin based tests for this are well established
(Dermal absorption in vitro skin test, OECD TG 428)
Skin irritation/
corrosion
Measures extent to which
the substance will irritate
and damage the skin
Substance is rubbed into the
shaved backs of rabbits and
they are killed 2 weeks later
(OECD TG 405). Tends to over
predict
Reconstituted human skin models are now accepted
and can be used in most cases (in vitro skin
corrosion and irritation tests, OECD TG 431 and 439)
Eye irritation/corrosion
Measures extent to which
the substance will irritate
the eyes
Substance is placed into the
eyes of live rabbits who are
monitored for up to 3 weeks
(OECD TG 404). Notoriously
unreliable test
Eyes from hens and cattle killed for food can now
be used to detect non-irritants and severe irritants
(BCOP and ICE ex vivo eye models, OECD TG 437 and
438). Detection of mild irritation can be assessed
using a combination of these tests and human
corneal epithelial models (currently undergoing
OECD acceptance)
Skin sensitisation
Measures the likelihood
that the substance will
cause an allergic reaction if
applied to the skin
The substance is rubbed onto
the shaved skin of guinea pigs
who are subjectively assessed
for allergy (Buehler or GPMT
test, OECD TG 406) or painted
onto the ears of mice who are
killed 6 days later to assess the
immune response (LLNA test,
OECD TG 429, 442a/b). The
more modern test, the LLNA,
predicts human reactions only
72% of the time
Several in vitro tests have been validated in
the EU; the peptide reactivity (DPRA) test which
measures the binding of the substance to proteins,
the keratinocyte assay and the human Cell Line
Activation Test (hCLAT) based on human skin cells
(all now currently undergoing OECD acceptance).
A testing strategy using these methods is already
being used by companies and is under discussion
at the OECD
Mutagenicity/
genotoxicity
Assesses the likelihood
cause genetic damage
which could lead to cancer
The substance is force-fed or
injected into mice or rats for
14 days who are then killed to
look at the effects on their cells
(OECD TG 474, 475, 486, 488)
A battery of two or three cell based tests is always
carried out before conducting an animal test
(Bacterial Reverse Mutation Test, OECD 471, in vitro
Mammalian Chromosome Aberration Test, OECD
473, in vitro Mammalian Cell Gene Mutation Test,
OECD TG 476, in vitro Mammalian Cell Micronucleus
Test, OECD 487). Positives should be assumed
genotoxic to avoid in vivo follow up
Repeated dose
Measures the effects of
repeated exposure to the
substance over a long
period
Rats (occasionally rabbits, mice
or even dogs) are force-fed,
forced to inhale or have the
substance rubbed onto their
shaved skin every day for 28
or 90 days before being killed
(OECD TGs 407-413). The ability
to correctly predict human
reactions (to drugs) using this
test is no more than 60%
In many cases can be avoided by ‘read across’ if the
exposure to the substance is likely to be extremely
low (TTC concept)

Skin absorption
Endpoint: The skin absorption safety test measures the extent to which
the substance will penetrate the skin.
Animal test: The substance is rubbed onto the shaved backs of rats
and they are killed the next day (OECD TG 427).
Alternative: Dermal absorption in vitro skin test (OECD TG 428).
Determining the extent to which the substance will absorb through
the skin and become systemically available is an important step in
being able to determine the Margin of Safety (MoS) of an ingredient,
and therefore the risk assessment of the product as a whole. In vitro
tests for skin absorption are well established and were one of the
first alternatives to be approved by the OECD in 2004. They measure
the extent of absorption of the substance through discs of donated
human skin into a fluid reservoir. These tests were shown to accurately
reproduce the same absorption through in vivo skin in the 1980s 12
and
were accepted for use in the EU in 199913
.
It is known that absorption through rodent skin tends to be higher
than it is in humans and therefore a rodent skin absorption study will
overestimate the extent to which the substance will penetrate human
skin by a factor of three14
. This is due to differences in skin thickness,
hair follicles and also immune responses. In vitro skin absorption
methods have the distinct advantage that human skin can be used.
The cost of in vivo and in vitro tests may be comparable due to the fact
that the substance is usually radio labelled to enable detection of the
substance.
Standard toxicity tests
—
12 Bronaugh, R. L. et al. 1982. Methods for in vitro percutaneous absorption studies
I: Comparison with in vivo results. Toxicol. Appl. Pharmacol. 62, 474 – 480.
13 SCCNFP 1999. Basic criteria for the in vitro assessment of percutaneous
absorption of cosmetic ingredients. Final guideline adopted by the SCCNFP,
23 June 1999, SCCNFP/0167/99.
14 Poet, T.S. 2000. Assessing dermal absorption. Toxicol. Sci. 58, 1 – 2.

Skin irritation/corrosion
Endpoint: Measures extent to which the substance will
irritate and damage the skin.
Animal test: The substance is rubbed into the shaved
backs of rabbits and they are killed two weeks later
(OECD TG 405).
Alternative: Reconstituted human epithelial (RhE) skin
models (OECD TG 431 and 439).
In vitro models based on reconstituted human epithelial
(RhE) skin have been developed since the 1980s. These
models comprise of small discs of cells grown into an
epidermal layer from human skin donated as waste from
cosmetic surgery. The models have now been thoroughly
validated and internationally approved by the OECD. The
tests can be used to classify substances as corrosive
(UN GHS category 1; some tests can be used for sub
classifications of this category), irritating (UN GHS category
2) and not irritating (not classified). The methods have a
wide applicability domain so there are only very limited
cases where an animal test could now be used for this
endpoint.
Skin corrosion can be assessed using RhE skin corrosion
model OECD TG 431 or other in vitro models, TG 430
(Corrositex®
) or TG 435 (TER). If the test is negative, the
RhE skin irritation models (OECD TG 439) should then be
used to assess if the substance is irritating or to confirm
that it is not irritating. Sometimes companies test their
substances using the skin irritation models (OECD TG 439)
only since cosmetics ingredients are not usually expected
to be corrosive. All OECD TG 439 methods predicted skin
irritation to at least 75% accuracy in the validation study15
,
although follow-up studies have shown they are actually
more accurate than this; for example in a study using 184
cosmetics, EpiSkin®
demonstrated 86% accuracy16
. Studies
show that the methods are more accurate and effective
than the Draize rabbit test they replace. For example,
a study has confirmed that the rabbit test tends to over
predict human skin reactions; Epiderm®
was found to be
76% accurate at predicting human skin patch test results
whereas the rabbit test was only correct 60% of the time 17
.
The test is so easy to conduct that these reconstituted
human epithelial (RhH) skin models can be purchased as
kits from the manufacturers: www.matek.com (EpiDerm®
),
www.skinethic.com (EpiSkin®
, Skin Ethic RHE®
) and
www.cellsytems.de (epiCS®
). Many contract testing
facilities are now familiar with the methods and will
use them as well. Contract testing facilities charge
approximately the same as for the rabbit test, but the kits
obtained directly from the manufacturers can be cheaper.
—
15 ESAC Statement on the scientific validity of in-vitro tests for skin irritation testing.
5th November 2008, see http://ecvam.jrc.it/
16 Cotovio, J. et al. 2007. In vitro acute skin irritancy of chemicals using the
validated EPISKIN model in a tiered strategy: Results and performance with 184
cosmetic ingredients. AATEX 14, Special Issue, 351 – 8.

17 Jirova, D. et al. 2007. Comparison of human skin irritation and photo-irritation
patch test data with cellular in vitro assays and animal in vivo data. AATEX 14,
special issue, 359 – 365.
Image supplied by IIVS

Eye irritation/corrosion
Endpoint: Tests for eye irritation and corrosion measure
the extent to which the substance will irritate the eyes if it
is accidentally spilt.
Animal test: The substance is placed into the eyes of
live rabbits who are monitored for up to three weeks
(OECD TG 404).
Alternative: BCOP and ICE ex vivo eye models
(OECD TG 437 and TG 438, HCE models, OECD TG in prep).
Isolated eyes from cattle or chickens killed for food
purposes can now be used to detect both severely
irritating/corrosive (GHS cat 1) and non-irritating
substances (not classified). The OECD TGs were approved
in 2009, and updated in 2013 to reflect the fact that they
can actually be used safely to detect non-irritants (non
classified substances). These ex vivo methods tend to over
predict the rabbit test results, which means they always
detect severely irritating substances but they may also
predict that a substance is irritating when it is not. These
methods can be used in a so-called top down/bottom
up approach; method A is used first if it is suspected
the substance is severely irritating (Cat 1) whilst method
B is used first if it is suspected that the substance is not
irritating (not classified). Determination of substances
that are irritating (Cat 2) can be achieved by testing both
methods and assuming irritancy if there is disagreement 18
.
Contract testing facilities charge approximately the
same to conduct the ex vivo tests as they do the rabbit
test. The rabbit test is notoriously cruel and unreliable,
with laboratories often giving very different results 19
and with only low to moderate correlation with human
responses as rabbits tend to experience more severe
effects than humans 20
.
Two methods based on reconstituted human corneal
epithelium are now being drafted as OECD Test Guidelines
to cover the eye irritation aspect. Final reports of the
Cosmetics Europe/ECVAM validation of two methods
(EpiOcular™ from www.mattek.com and SkinEthic™
Human Reconstructed Corneal Epithelium (HCE) from
www.skinethic.com) are expected in 2014. Multi-
laboratory test results have already been published
showing that there is over 95% agreement in test results
between laboratories for both EpiOcular® 21
and HCE® 22
.
An assessment of 435 cosmetics substances has shown
that SkinEthic HCE®
is 82% accurate 23
and a study by
BASF®
found EpiOcular to be over 85% accurate 24
.
The methods are available from the manufacturers
and are already being used by companies to screen
their substances as part of this top-down/ bottom-up
approach.
There are other methods available that have a more
limited applicability but that can also be used in
conjunction with other test methods: the Fluorescein
Leakage Test Method uses an animal-derived epithelial
cell line monolayer that can identify severe eye irritants
(OECD TG 460, accepted 2012), and the Short Time Exposure
(STE) and the Cytosensor Microphysiometer (CM) tests are
also based on animal cell lines but can detect both severe
irritants and non-irritants (both draft OECD TGs).
—
18 Scott, L. et al. 2010. A proposed eye irritation testing strategy to reduce and
replace in vivo studies using Bottom-Up and Top-Down approaches. Toxicol. in
Vitro 24:1-9.
19 Ohno, Y. Et al. 1999. Interlaboratory validation of the in vitro eye irritation tests
for cosmetic ingredients. (1) Overview of the validation study and Draize scores
for the evaluation of the tests. Toxicol. In Vitro. 13, 73 – 98. And Lordo, R.A., et
al. 1999. Comparing and evaluating alternative (in vitro) tests on their ability to
predict the Draize maximum average score. Toxicol. In Vitro. 13, 45 – 72. And
Weil, C.S. and Scala, R.A. 1971. Study of intra – and interlaboratory variability
in the results of rabbit eye and skin irritation tests. Toxicol. Appl. Pharm. 19,
276 – 360.
20 Freeberg, F.E. et al. 1986. Human and rabbit eye responses to chemical insult.
Fundam. Appl. Toxicol. 7, 626 – 634.
21 Pfannenbecker, U. Et al. 2013. Cosmetics Europe multi-laboratory pre-validation
of the Epiocular™ reconstituted human tissue test method for the prediction of
eye irritation. Toxicol. in Vitro 27, 619–626.
22 Alepee, N. et al. 2013. Cosmetics Europe multi-laboratory pre-validation of
the Skinethic™ reconstituted human corneal epithelium test method for the
prediction of eye irritation. Toxicol In Vitro 27, 1476 – 88.
23 Cotovio, J. et al. 2010. In vitro assessment of eye irritancy using the
Reconstructed Human Corneal Epithelial SkinEthic™ HCE model: Application
to 435 substances from consumer products industry. Toxicology in Vitro 24
523–537.
24 Kolle SN. et al. 2011. In-house validation of the EpiOcular(TM) eye irritation test
and its combination with the bovine corneal opacity and permeability test for
the assessment of ocular irritation. Altern Lab Anim. 39, 365 – 87.

—
25 OECD 2012. The Adverse Outcome Pathway for Skin Sensitisation Initiated by
Covalent Binding to Proteins. Part 1 and 2. Series on Testing and Assessment
No. 168.
26 EURL-ECVAM (2013) EURL ECVAM Recommendation on the Direct Peptide
Reactivity Assay (DPRA) for Skin Sensitisation Testing. EUR 26383 EN
27 Ahlfors, S. R. et al. 2003. Reactivity of contact allergenic haptens to amino acid
residues in a model carrier peptide, and characterization of formed peptide-
hapten adducts. Skin Pharmacol. Appl. Skin Physiol. 16, 59 – 68.
28 Gerberick, G. F. et al. 2007. Quantification of chemical peptide reactivity for
screening contact allergens: a classification tree model approach. Toxicol. Sci.
97, 417 – 427.
29 Stokes, W. et al. 2012. Comparison of the DPRA with a three-test battery for in
vitro evaluation of skin sensitization. NICEATM-ICCVAM SOT 2012 Poster
30 Bauch, A. et al. 2011. Intralaboratory validation of four in vitro assays for the
prediction of the skin sensitizing potential of chemicals. Toxicol. in Vitro 6,
1162 – 1168.
31 Natsch, A. et al. 2013. A dataset on 145 chemicals tested in alternative assays
for skin sensitization undergoing prevalidation. Journal of Applied Toxicology,
doi: 10.1002/jat.2868.[epub ahead of print]
32 EURL ECVAM (2013) Draft recommendation on the KeratinoSensTM assay for skin
sensitisation testing.
33 Bauch, A. et al. 2012. Putting the parts together: combining in vitro methods to
test for skin sensitizing potentials. Regul. Toxicol. Pharmacol. 63, 489 – 504.
34 Natsch, A. et al. 2013.
35 Ashikaga, T. et al. 2010. A comparative evaluation of in vitro skin sensitisation
tests: the human cell-line activation test (hCLAT) versus the local lymph node
assay (LLNA). Altern. Lab. Anim. 38, 275 – 84.
36 Natsch, A. et al. 2013 and Bauch, A. et al. 2011 and Bauch, A. et al. 2012.
37 Nastch, A. et al. 2013.
Skin sensitisation
Endpoint: Skin sensitisation is an allergic reaction to a
particular substance that results in the development
of skin inflammation and itchiness. The skin becomes
increasingly reactive to the substance each time it is
exposed to it.
Animal test: The substance is rubbed onto the shaved
skin of guinea pigs who are subjectively assessed for
allergy (Buehler or GPMT test, OECD TG 406), or painted
onto the ears of mice who are killed six days later to
assess the immune response (LLNA test, OECD TG 429,
442a/b).
Alternative: DPRA, KeratinoSens®
and h-CLAT in a testing
strategy (OECD testing strategy in preparation).
The mechanism of how skin reacts to sensitising
substances to produce an allergic reaction is well
understood 25
. A key early step is the reaction of proteins in
the skin to the substance, a process called ‘haptenation’.
This can be measured using peptide reactivity tests which
measure depletion of cysteine or lysine-based peptides
following 24 hours incubation with the test substance.
One of these protein reactivity tests (the Direct Peptide
Reactivity Assay – DPRA) has been used by industry since
the early 2000s and completed ECVAM pre-validation
in 2013. ECVAM concluded that the test could accurately
distinguish sensitisers from non-sensitisers 82% of the
time 26
. Previous industry studies gave similar results of
94% 27
, 89% 28
, 85% 29
, 91% 30
and 80% 31
.
ECVAM is also due to publish its recommendation in 2014
on two other skin cell based tests that can be used in
conjunction with the DPRA; KeratinoSens®
and h-CLAT
(human cell line activation test). These are based on
human cell lines and measure the activation of genes
known to be involved in triggering the immune response.
ECVAM found that the accuracy of the KeratinoSens®
to
discriminate skin sensitisers from non-sensitisers was
90% 32
. These figures are similar to those published by
the industry in two additional studies giving accuracy
compared to the LLNA of 81% 33
and 77% 34
. Studies using
the h-CLAT by cosmetics company Shiseido show 84%
agreement on 100 chemicals 35
. A further test using a
human lymphoma (immune) cell line (Modified myeloid
U937 skin sensitisation test (mMUSST)) has also been
developed and tested by companies 36
.
The OECD is working on all three Test Guidelines, and
an integrated testing strategy for skin sensitisation
using these methods is expected to be published by
2015. Companies are already using these methods in
combination however. A study by BASF showed that a
combination of two out of three tests gave a reported
accuracy of 94% compared to human data, and a Proctor
and Gamble/Givaudan study found a correlation of 81%
compared to LLNA (animal test) results 37
. These tests
can be used in the EU for regulatory purposes since only
simple classification of sensitisation (yes or no) is required
under EU chemicals REACH legislation. The LLNA can also
only give this classification and only has an accuracy of
72% (when compared to human data) with a risk of both

—
38 European Commission. (2000). Opinion on the murine local lymph node assay
(LLNA) adopted by the SCCNFP during the 12th plenary meeting of 3 May 2000.
39 Bauch, A. et al. 2012.
40 Chaundry, Q. et al. 2010. Global QSAR models of skin sensitisers for regulatory
purposes. Chem. Central J. 4, S5.
false positive and negative results 38
. Studies comparing
the new test methods with known human skin allergens
show that the in vitro tests are more accurate than this.
For example, one study showed the accuracy of the DPRA
to be 86% and the KeratinoSens®
80% 39
. The three in vitro
tests can be carried out for the same price as the LLNA
animal test (around 4,000 Euros). The DPRA takes one day
to run, and the KeratinoSens®
takes four days, whilst the
LLNA takes six days for the animal part of the test only.
QSAR (Quantitative Structure-Activity Relationship)
computer models have particularly strong predictive
strength for skin sensitisation and give results even
more quickly and cheaply. They are based on datasets
of known chemicals with known results for the endpoint
of interest. By inputting the chemical structure of a new
chemical they can predict its similarity to other chemicals
in the database and therefore the likely toxicity. QSARS
work well for the skin sensitisation endpoint because skin
reactivity can be predicted based on chemical structure
alone. Models include DEREK, TOPKAT, TOPS-MODE,
CAESAR and the OECD Toolbox. Several models have
given accurate results compared to known data, for
example, CAESAR made 90% correct predictions on 42
chemicals 40
, see http://www.antares-life.eu/ for lists of
models.
“The DPRA takes one
day to run, and the
KeratinoSens®
takes
four days, whilst the
LLNA takes six days for
the animal part of the
test only.”

—
41 Kirkland, D. M. et al. 2005. Evaluation of the ability of a battery of three in vitro
genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I:
Sensitivity, specificity and relative predictivity. Mut. Res. 7, 70.
42 Fowler P, et al. 2012. Reduction of misleading (“false”) positive results in
mammalian cell genotoxicity assays. I. Choice of cell type. Mut. Res. 742, 11 – 25.
And Fowler P, et al. 2012. Reduction of misleading (“false”) positive results
in mammalian cell genotoxicity assays. II. Importance of accurate toxicity
measurement. Mut. Res. 747, 104 – 17.

43 Kirkland D, et al. 2011. A core in vitro genotoxicity battery comprising the Ames
test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens
and in vivo genotoxins. Mutat Res 721, 27 – 73. And EFSA, 2011. Scientific Opinion
of the Scientific Committee on genotoxicity testing strategies applicable to
food and feed safety assessment. EFSA Journal 2011; 9(9):2379 (69pp) and
COM, 2011. Guidance on a Strategy for Testing of Chemicals for Mutagenicity.
Committee on Mutagenicity of Chemicals in Food, Consumer Products and the
Environment (COM). Department of Health, London. [http://www.iacom.org.uk/
guidstate/documents/COMGuidanceFINAL2.pdf]
44 Hu T, et al. 2010. Xenobiotic metabolism gene expression in the EpiDerm™ in
vitro 3D human
Mutagenicity/genotoxicity
Endpoint: The mutagenicity/genotoxicity safety test assesses the
likelihood that the substance will cause genetic damage which could
lead to cancer.
Animal test: The substance is force-fed or injected into mice or rats for
14 days who are then killed to look at the effects on their cells (OECD
TG 474, 475, 486, 488).
Alternative: Bacterial Reverse Mutation Assay (Ames test) (OECD
TG 471), In Vitro Mammalian Cell Gene Mutation Test (OECD TG 476),
In Vitro Mammalian Chromosome Aberration Test (OECD TG 473),
In Vitro Mammalian Cell Micronucleus Test (OECD TG 487).
Mutagenicity/genotoxicity is always assessed initially in vitro using
bacterial and other cell based tests. These tests assess the extent of
damage to the chromosomes (containing genes) in the cells that could
be indicative that the substance causes cancer. In many cases it is
possible to determine whether a substance is likely to be genotoxic
by conducting up to three of these cell based tests, covering effects on
gene mutation (TG 471 and TG 476), changes to chromosome structure
(TG 473) and number (TG 487). In combination these tests have been
shown to be 85 – 90% predictive of rodent carcinogenicity test results
across a large number of chemicals41
.
These in vitro tests are often accused of being too protective,
i.e. safe chemicals can be mistakenly predicted to be genotoxic.
However this is inconclusive as the results are always compared to
tests in rats and mice rather than humans. The common approach is
to ‘follow up’ these positive results using an in vivo mouse or rat test.
However, follow up of positive results can be avoided by careful choice
of cell type (human cells being preferable), dose levels and method
of assessment of the damage42
. Cells should be exposed to the test
substance in the presence and absence of an appropriate metabolic
activation system. It has recently been recommended in Europe
that only two in vitro tests are required if the newest test, the In Vitro
Mammalian Cell Micronucleus Test (TG 487), is used because it looks
at changes to both chromosome structure and number43
. The use of
RhE models (see Skin irritation/corrosion) is currently being examined
by Cosmetics Europe to see if this adds to the assessment, especially
since they use human tissue44
.

—
45 Olson, H. et al. 2000. Concordance of the toxicity of pharmaceuticals in humans
and in animals. Reg. Toxicol. Pharmacol. 32, 56 – 67. And Spanhaak, S. et al.
2008. Species concordance for liver injury from the safety intelligence program
board. Cambridge, UK: BioWisdom, Ltd. : http://www.biowisdom.com/files/
SIP_Board_Species_Concordance.pdf (accessed 24 August 2009).
46 Kroes, R. et al. 2007. Application of the threshold of toxicological concern
(TTC) to the safety evaluation of cosmetic ingredients. Food Chem. Toxicol. 45,
2533 – 2562.

47 Scientific Committee on Consumer Safety (SCCS), Scientific Committee on Health
and Environmental Risks (SCHER), Scientific Committee on Emerging and Newly
Identified Health Risks, (SCENIHR). 2012. OPINION ON Use of the Threshold
of Toxicological Concern (TTC) Approach for Human Safety Assessment of
Chemical Substances with focus on Cosmetics and Consumer Products.
European Commission, SCCP/1171/08. http://www.oecd.org/env/ehs/risk-
assessment/groupingofchemicalschemicalcategoriesandread-across.htm
48 http://www.oecd.org/env/ehs/risk-assessment/groupingofchemicalschemicalc
ategoriesandread-across.htm
Repeated dose
Endpoint: Measures the effects of repeated exposure to
the substance over a period of time.
Animal test: Rats (occasionally rabbits, mice or even dogs)
are force-fed, forced to inhale or have the substance
rubbed onto their shaved skin every day for 28 or 90 days
before being killed (OECD TGs 407 – 413).
Alternative: Until a testing strategy using in vitro tests
is developed and validated, repeated dose testing can
often be avoided through the use of the TTC (threshold
of toxicological concern) approach and/or read across
approaches.
Several reviews of the ability of rodent tests to predict
human toxicity, mainly in the area of pharmaceuticals,
have found that they are only about 40 – 60% predictive 45
.
Nonetheless, repeated dose information is often required
for new cosmetics ingredients in order to obtain the No
Observed Adverse Effect Level (NOAEL) to perform the risk
assessment. Due to the fact that cosmetics substances
are often used in such low quantities, in many cases
animal tests can be avoided by use of the TTC concept.
As the calculations are necessarily conservative, this
concept could mitigate the perceived need for animal
tests for a great many cosmetic ingredients and provide
the required protection to consumers.
The TTC approach is based on the concept that for all
substances, there is a level of exposure below which
there is hardly any risk to human health, regardless of
how toxic the substance is. If the exposure of a substance
in a cosmetics product is known (which it should be as
part of the risk assessment, see Product Testing), and if it
is very low, then even if the substance is assumed to be
toxic, testing will not affect the safety of the product and
the TTC approach could apply. Instead of conducting an
animal test, the risk assessor will do an evaluation (based
on chemical structural similarity to other substances) as
to the likely toxicity class of the substance, followed by a
calculation of maximum daily exposure. If the substance
falls below a certain value then it can be considered
‘safe’. The TTC concept was first used for food additives,
but research by the cosmetics industry has shown it to be
relevant for cosmetics 46
and examples and databases are
now available 47
. Our calculations show that the concept
could be used for antioxidants, UV filters, chelating
agents, foam stabilisers, thickeners, preservatives,
humectants, pearlescing/opacifying agents, fragrances
and, if concentrations are kept to a certain level, also
pigments and dyes. Across a range of cosmetics products
these constitute 67% (two-thirds) of the ingredients within
a product.
Read across or category approaches can also be used
when there is existing data on a structurally similar
substance(s). Substances whose physicochemical,
toxicological and ecotoxicological properties are likely
to be similar or follow a regular pattern as a result of
structural similarity may be considered as a group, or
‘category’ of substances. In this case, existing data on
one or more members of the group can be used to
provide data (to read across) for the other members, and
new testing can be avoided. See OECD Guidance on the
Grouping of Chemicals and the use of the OECD Toolbox 48
.
Companies innovating by modifying substances slightly to
improve them may well find that they are justified in using
read across from the original substance instead of
animal testing.
A variety of cell-based models are available that either
use long-lasting liver cells or incorporate a range of cell
types into a ‘microchip’. These are currently used to screen
substances for long term toxicity but do not yet have
regulatory acceptance.

Table 2:
Non-standard cosmetics toxicity tests and the available options to avoid animal testing
Endpoint Tests for Animal test
Options to avoid
animal test
Phototoxicity
Whether the substance will
cause a reaction if applied
to the skin and the skin is
then exposed to sunlight
No suitable animal test exists,
the in vitro test is the standard
test
Not always required. Cell based tests have been in
place for some time (3T3 NRU cell-based test, OECD
TG 432), negative results can be confirmed in human
skin tests (in vivo or in vitro)
Acute toxicity
Assesses the amount of
the substance that will
cause severe toxic effects
if accidentally ingested,
inhaled or rubbed on the
skin
Rats are exposed to a very
high dose of the substance
such that a number of them
are expected to die (OECD TG
402, 403, 420, 423, 425, 436)
Not always required because assessing repeated
dose toxicity is considered more useful.
Cell based tests such as the NRU3T3 can be used
to predict lack of toxicity very accurately (ECVAM
recommendation 2013)
Carcinogenicity
that a substance will
cause cancer if people are
exposed to it over a long
period
Rats or mice are fed the
substance for two years to see
if they get cancer (OECD TG
451, 452). Costs $2 million and
only predicts human cancer
42% of the time
Rarely a regulatory requirement. Rarely conducted
because it takes so long and is so unreliable.
Companies use the genotoxicity tests above and
assume if the substance is genotoxic then it may
also cause cancer. Cell transformation assays (CTA)
have been in use for 50 years (EU Test Method B.21),
predict 90% of known human carcinogens and
can be used for follow up if necessary (currently
undergoing OECD acceptance)
Reproductive toxicity
reduce fertility or cause
developmental problems
to the fetus
Pregnant female rabbits or rats
are force-fed the substance
and then killed along with their
unborn babies (OECD TG 414).
Such tests take a long time
and use hundreds of animals.
Studies have shown they only
detect 60% of known human
toxicants
Rarely a regulatory requirement. In many cases can
be avoided by ‘read across’ or if the exposure to the
substance is likely to be extremely low (TTC concept)
Endocrine disruption
interfere with the body’s
endocrine (hormone)
system producing harmful
effects
No single established animal
test for endocrine disruption
exists (and is unlikely to). The
Hershberger assay looks at
the effects on castrated male
rats who are injected with or
force-fed the substance for
10 days before being killed.
(OECD TG 441)
Not a regulatory requirement. Receptor binding
assays such as the Stably Transfected Transcriptional
Activation assay (STTA) (OECD TG 455) and the
BG1Luc Estrogen Receptor Transactivation Test
Method for Identifying Estrogen Receptor Agonists
and Antagonists (OECD TG 457) and the H295R
Steroidogenesis Assay (OECD TG 456) can be
used to screen for potential endocrine disrupting
properties
Toxicokinetics
Assesses how the body
deals with a substance, i.e.
whether it is metabolised
or not and how long it
stays in the body
Rabbits or rats are forced
to consume the substance
then are placed in cages on
their own before being killed
and their organs examined
(OECD TG 417). Poor estimates
based on animal studies are
responsible for 30% of drug
failures.
Rarely a regulatory requirement. Skin absorption
(OECD TG 428) and liver cell metabolism tests (see
OECD TG 417) can be put into a PBPK computer
model that combines information to predict what the
body will do.

Phototoxicity
Endpoint: The skin absorption safety test measures the extent to which
the substance will penetrate the skin.
Animal test: The photoxicity test measures the extent to which the
substance, if applied to the skin, might react with sunlight and become
more dangerous.
Alternative: Not always required; 3T3 NRU cell-based test
(OECD TG 432).
Information on whether a cosmetics ingredient is likely to cause
photo-induced toxicity is only required if the product is intended for
use on sunlight-exposed skin, for example face cream. The test is
used to check that there is not a reaction between the substance and
sunlight that makes it more toxic, usually more of an irritant. There is
no validated animal test for phototoxicity. In vitro tests for phototoxicity
have been in place for years; they were validated in the 1990s and
approved by the OECD in 2004. The NRU3T3 test (OECD TG 432) is
based on an animal cell line and measures the number of cells that
die when in contact with the substance and radiation.
This simple test has been accused of giving false positive results,
i.e. over predicting phototoxicity. However, a recent workshop on the
use of the test for drug products highlighted that companies need
to adhere to the OECD Test Guideline to ensure its correct use and
avoid the use of old cells or high doses 49
. In vitro test results can also
be followed up by carefully conducted tests in humans (see Product
Testing) or reconstituted human skin models (see Skin Irritation). As an
analogy, Sun Protection Factor (SPF) claims are tested using human
volunteers in a photo patch testing protocol 50
.
Non-standard
toxicity tests
—
49 Ceridono, M. et al. 2012. The 3T3 neutral red uptake phototoxicity test: Practical
experience and implications for phototoxicity testing – The report of an ECVAM–
EFPIA workshop. Regul. Toxicol. Pharmacol. 63, 480–488.
50 International Sun Protection Factor (SPF) Test Method (Colipa 2006)
“There is no
validated
animal test for
phototoxicity.”

Acute toxicity
Endpoint: The acute toxicity test assesses the amount of the substance
that will cause severe toxic effects if accidentally ingested, inhaled or
rubbed on the skin.
Animal test: Rats are exposed to a very high dose of the substance
such that a number of them are expected to die (OECD TG 402, 403,
420, 423, 425, 436).
Alternative: Not always required because assessing repeated dose
toxicity is considered more useful.
Single dose studies for cosmetics ingredients are not considered useful
because these tests were designed years ago as a crude measure
of the toxicity of chemicals. Today it is more common to see repeated
dose data instead of LD50 animal test information for cosmetics
ingredients since cosmetics are not expected to be very toxic and
repeated dose information is usually required in order to directly
determine the NOAEL. In the EU acute toxicity data is not insisted upon
if repeated dose information is available.
Cell based tests such as the NRU3T3 (see Photoxicity) can be used to
predict lack of toxicity very accurately. The OECD has issued Guidance
Document 129 which outlines the test and how it can be used to
estimate the starting dose for an animal test, following a review by US
authorities 51
. However, ECVAM has recently concluded in a large scale
analysis that shows that the test can be safely used to detect non-
toxic, non-classified substances (LD50 values greater than 2,000 mg/
kg bw/d) 52
. Only two substances (plant toxins) that were classified for
acute toxicity were not identified by the test, therefore if the test result
is negative it can be trusted. Since most substances are non-toxic 53
,
the use of this test can avoid further testing in most cases.
—
51 OECD (2010) Guidance document on using cytotoxicity tests to estimate starting
doses for acute oral systemic toxicity tests. Series on Testing and Assessment.
No. 129.
52 EURL ECVAM Recommendation on the 3T3 Neutral Red Uptake Cytotoxicity
Assay for Acute Oral Toxicity Testing, 2013. Report EUR 25946 EN
53 Bulgheroni A, et al. 2009. Estimation of acute oral toxicity using the No Observed
Adverse Effect Level (NOAEL) from the 28 day repeated dose toxicity studies in
rats. Regul Toxicol Pharmacol. 53, 16 – 9.

Carcinogenicity
Endpoint: A carcinogen is a substance that causes cancer or increases
the likelihood that someone will develop cancer.
Animal test: Rats or mice are fed the substance for two years to see if
they get cancer (OECD TG 451, 452).
Alternative: Very rarely conducted, carcinogenicity can be assumed
from genotoxicity tests or tested using the Cell Transformation Assays
(OECD TG in preparation).
The rat carcinogenicity bioassay is a notoriously unreliable study with
an estimated predictivity of only 42%54
. It is expensive (approximately
one million Euros) and time-consuming (two years minimum) and for
these reasons is almost never conducted for cosmetics substances55
. It
is even being phased out for pharmaceuticals56
. In practice, cosmetics
developers use the genotoxicity tests (see Genotoxicity) and assume
if the substance is genotoxic then it may cause cancer. Although this
may rule out some substances for future use that may be safe, it is
normal practice and protects consumers.
Follow up testing can be carried out using the Cell Transformation
Assays (CTA) using rodent cells (Syrian Hamster Embryo (SHE), Balb/
c3T3 and Bhas42 cells), which detects both genotoxic and non-
genotoxic carcinogens. These assays have been in use for over 40
years but have more recently been improved and validated. An OECD
review in 2007 concluded that 90 – 95% of human carcinogens could
be detected57
and a draft Test Guideline is near completion. The test
takes 3 – 6 weeks compared to over two years for the rat bioassay
and costs approximately 500 Euros per test compared to one million
Euros for the rat bioassay. In the meantime these assays have been
endorsed by ECVAM for their use in carcinogenicity testing58
and an
assessment of their use in an integrated testing strategy confirmed the
findings of the OECD that they can detect 90 – 95% of genotoxic and
non-genotoxic carcinogens59
.
—
54 Knight, A. et al. 2005. Which drugs cause cancer? Br. Med. J. USA 5, 477.
55 Adler, S. et al. 2011. Alternative (non-animal) methods for cosmetics testing:
current status and future prospects—2010. Arch Toxicol. 85, 367 – 485.
56 Sistare, F.D. et al. 2011. An analysis of pharmaceutical experience with decades
of rat carcinogenicity testing: support for a proposal to modify current regulatory
guidelines. Toxicol Pathol 39, 716 – 44.
57 OECD 2007. Detailed review paper on cell transformation assays for detection of
chemical carcinogens. Series on Testing and Assessment No 31.
See: www.oecd.org.
58 EURL ECVAM RECOMMENDATION on three Cell Transformation Assays using
Syrian Hamster Embryo Cells (SHE) and the BALB/c 3T3 Mouse Fibroblast Cell
Line for In Vitro Carcinogenicity Testing, 2012 and EURL ECVAM Recommendation
on the Cell Transformation Assay based on the Bhas 42 cell line, 2013.
59 Benigni, R. et al. 2013. In vitro cell transformation assays for an integrated,
alternative assessment of carcinogenicity: a data-based analysis. Mutagenesis,
28, 107 – 116.
“The rat
carcinogenicity
bioassay is
a notoriously
unreliable study
with an estimated
predictivity of only
42%”

Reproductive toxicity
Endpoint: Reproductive toxicity refers to a wide variety of
adverse effects that may occur in different phases within
the reproductive cycle, including effects on male and
female fertility, sexual behaviour, embryo implantation,
embryo development, birth and growth and development
of the young.
Animal test: Pregnant female rabbits or rats are force-fed
the substance and then killed along with their unborn
babies (OECD TG 414).
Alternative: Reproductive toxicity tests are not usually
a standard requirement. In some cases they can be
avoided through the use of read across or TTC. The
embryonic stem cell test (EST) can be used to screen
for developmental toxicity.
Tests for reproductive toxicity are not considered a core
requirement for cosmetics ingredients in Europe and
may only be conducted if “considerable oral intake or
dermal absorption is expected”60
. This is because in
many cases consumers will be exposed to such low
levels of the individual substances that reproductive
effects, even if the substance has the potential to cause
them, are very unlikely to occur. Again, the TTC approach,
whose feasibility for reproduction endpoints has been
demonstrated for chemicals generally61
, can also be used
(see Repeated Dose). Read across and QSARs can also be
used for this endpoint (see Repeated Dose).
Those companies that voluntarily undertake reproductive
toxicity tests usually only carry out the developmental
toxicity test62
. This test takes at least four weeks, uses
hundreds of animals and costs over 60,000 Euros. In
addition, a number of studies have shown that it only
detects about 60% of known human reproductive
toxicants63, 64
. An in vitro test using animal-based stem
cells has been developed, however, to screen for harmful
effects on the developing fetus. The embryonic stem cell
test (EST) takes advantage of the nature of stem cells to
use failure to differentiate into beating heart muscles as
an indication of the developmental toxicity potential of a
chemical.
The EST was fully validated by ECVAM in 2002 and shown
to have an overall accuracy of 78% with 20 substances 65
.
Although not yet accepted for regulatory purposes the EST
is used by industry for in-house screening purposes. In
2008, Pfizer concluded that the overall performance of the
EST was generally good with an accuracy of 75% for 63
chemicals, and that they were confident to use the assay
to aid compound-development decisions66
. Improvements
have been made recently to increase applicability 67
and
speed of the assay68
and to account for metabolism69
.
The test takes only 10 days to conduct and costs
approximately 3,000 Euros.
Researchers are also working on a battery of in vitro
tests that can cover the entire reproductive cycle. The EU
ReProTect project has recently concluded that a battery
of ten cell tests, including those described here, “allowed
a robust prediction of adverse effects on fertility and
embryonic development”70
.
—
60 SCCS 2012. The SCCS’s Notes of Guidance for the Testing of Cosmetic Substances
and their Safety Evaluation, 8th Revision SCCS/1501/12.
61 Bernauer, U. et al. 2008. Exposure-triggered reproductive toxicity testing under
the REACH legislation: A proposal to define significant/relevant exposure.
Toxicol. Lett. 176, 68 – 76.
62 Rogiers, W. and Pauwels, M. 2008. Safety assessment of cosmetics in Europe.
Curr. Prob. Dermatol. 36. Karger; Basel, Switzerland.
63 Hurtt, M. E. et al. 2003. Proposal for a tiered approach to developmental toxicity
testing for veterinary pharmaceutical products for food producing animals. Food
Chem. Toxicol. 41, 611 – 619.
64 Bailey, J. et al. 2005. The future of teratology research is in vitro. Biogenic
Amines 19, 97 – 145.
65 Genschow E, Spielmann H, Scholz G, Pohl I, Seiler A, Clemann N, Bremer S,
Becker K. Validation of the embryonic stem cell test in the international ECVAM
validation study on three in vitro embryotoxicity tests. Altern Lab Anim. 2004
Sep;32(3):209 – 44.
66 Paquette, J. A. et al. 2008. Assessment of the embryonic stem cell test and
application and use in the pharmaceutical industry. Birth Defects Res. B Dev.
Repro. Toxicol. 83, 104 – 111.
67 Dartel, D. A. M. et al. 2010. Monitoring Developmental Toxicity in the Embryonic
Stem Cell Test Using Differential Gene Expression of Differentiation-Related
Genes. Toxicol. Sci. 116, 130 – 139.
68 Peters, A. K. et al. 2008. Evaluation of the embryotoxic potency of compounds
in a newly revised high throughput embryonic stem cell test. Toxicol Sci. 105,
342 – 350.
69 Hettwer, M. et al. 2010. Metabolic activation capacity by primary hepatocytes
expand the applicability of the embryonic stem cell test as an alternative to
experimental animal testing. Reprod. Toxicol. 30, 13 – 20.
70 Schenk, B. et al. 2010. The ReProTect feasibility study, a novel comprehensive in
vitro approach to detect reproductive toxicants. Reprod. Toxicol. 30, 200 – 218.

Endocrine disruption
Endpoint: Tests seek to assess the likelihood that the substance will
interfere with the body’s endocrine (hormone) system producing
harmful effects.
Animal test: No single established animal test for endocrine disruption
exists (and is unlikely to). The Hershberger assay looks at the
effects on castrated male rats who are injected with or force-fed the
substance for 10 days before being killed (OECD TG 441).
Alternative: Not a standard endpoint, receptor binding assays
(e.g. OECD TG 455, 456 and 457) can help screen.
Although there is much scientific and regulatory interest in the
potential for substances to be endocrine disruptors, there are no
standard animal or non-animal tests for this endpoint; there is even
disagreement about the point at which a substance can be considered
an endocrine disruptor.
Nonetheless, there are now a range of receptor binding assays
that can be used to screen cosmetics ingredients for potential endocrine
(hormone) disrupting properties. These assays work by using a labelled
compound, that when it binds to a receptor can be used to detect that
receptor. The extent to which the labelled compound can be detected
in the presence of the test substance gives a measure of how much
the substance has interfered with the receptors related to hormone
production. ECVAM and the OECD are in the process of validating a
range of these. Already there is the Stably Transfected Transcriptional
Activation assay (STTA) (OECD TG 455), the H29SR Steroidogenesis
Assay (OECD TG 456) and the BG1Luc estrogen receptor transactivation
test method for identifying estrogen receptor agonists and antagonists
(OECD TG 457). There are more tests in development and validation
that cover the male hormones (androgens).
“Hershberger assay
looks at the effects
on castrated
male rats who
are injected with
or force-fed the
substance for 10
days before being
killed”

Toxicokinetics
Endpoint: Toxicokinetics is an assessment of how the body deals with
a substance, i.e. whether it is metabolised or not and how long it stays
in the body, which helps to aid decision on the safety of the substance.
Animal test: Rabbits or rats are forced to consume the substance then
are placed in cages on their own before being killed and their organs
examined (OECD TG 417).
Alternative: Toxicokinetic studies are rarely a legal requirement for the
safety assessment of cosmetics. The use of pharmacokinetic computer
models, together with in vitro dermal absorption and metabolism
data, can adequately replace the key components.
It is usually not mandatory to have animal-based toxicokinetics data.
In a review of EU cosmetics dossiers, less than 50% of dossiers had
toxicokinetic data and the regulator did not request the conduct of an
in vivo test71
. However, toxicokinetic data, or aspects of it, can help
in the risk assessment. The skin is the main route for the absorption
of cosmetics and can already be modelled using the regulatory
approved in vitro skin absorption method (see Skin Absorption).
Metabolism can be predicted through the use of high-throughput
assays on cultured human hepatocytes (liver cells). Results from these
tests can then be run through computer generated physiologically-
based toxicokinetic models (PBTK) to predict the distribution and
excretion of substances through the human body. These have been
used by the pharmaceutical industry with growing sophistication since
the 1970s72
and a number of studies have demonstrated their high
prediction rate 73
. In fact, before in vitro studies on human cell models
were routinely used by the pharmaceutical industry, the failure rate of
drugs in clinical trials due to poor prediction of pharmacokinetics was
40% 74
– now it is only 10% 75
. There are companies who offer this as
a service. A recent study showed that in vitro liver cell tests with PBPK
modelling gave better prediction accuracy for humans compared to
in vivo rat and dog tests 76
. The option to use computer models and in
vitro assays on liver cells to address metabolism has been included in
the recently updated OECD TG 417 on toxicokinetics.
—
71 Pauwels, M. et al. 2009. Critical analysis of the SCCNFP/SCCP safety assessment
of cosmetic ingredients (2000 – 2006) Food Chem. Toxicol. 47, 898 – 905.
72 Andersen, M. E. 2003. Toxicokinetic modeling and its applications in chemical
risk assessment; Toxicol. Lett. 138, 9 – 27.
73 Poulin, P. and Theil, F. P. 2002. Predictions of pharmacokinetics prior to in vivo
studies I: Mechanism based prediction of volume of distribution. J. Pharm Sci,
91, 129 – 156. And Jones, H. M. et al. 2006. A novel strategy for physiologically-
based predictions of human pharmacokinetics. Clin. Pharmacokinet. 45,
511 – 542. And Kusama, M. et al. 2010. In silico classification of major clearance
pathways of drugs based on physicochemical parameters. Drug Metab.
Dispos. 38, 1362 – 1370
74 Kola, I. and Landis, J. 2004. Can the pharmaceutical industry reduce attrition
rates? Nature Rev. 3, 711 – 715.
75 McKim, J. M. Jr. 2010. Building a tiered approach to in vitro predictive toxicity
screening: A focus on assays with in vivo relevance. Combinat. Chem. High
Throughput Screen. 13, 188 – 206.
76 Yamazaki, S. et al. 2011. Prediction of oral pharmacokinetics of cMet kinase
inhibitors in humans: Physiologically-based pharmacokinetic model versus
traditional one compartment model. Drug Metab. Dispos. vol. 39, 383 – 393

—
79 In China, ‘ordinary cosmetics’ include hair care, nail care, skin care, fragrances,
make-up, and nail/toe cosmetics.
Over 80% of the world still allows animal testing for cosmetics.
Yet the animal tests that have traditionally been used to test the
safety of cosmetics are cruel, unnecessary, expensive and unreliable.
As demonstrated in this report, quicker, cheaper and more reliable
modern alternatives have been validated, can be used by companies
and should be accepted by regulators worldwide.
The public wants to see an end to the suffering of animals used to test
cosmetics and personal care products. With better alternatives now
available and becoming ever more sophisticated, governments can
respond to public opinion and make a decision to end animal testing
for cosmetics whilst also providing better safety of these products.
The European Union’s trailblazing 2013 ban set a humane example to
the world, and has demonstrated that it is possible to have a vibrant,
innovative and profitable cosmetics market without the use of animal
tests. Other countries are following suit, with bans in Israel and India,
and positive developments across Asia, including China which will end
its animal testing requirement for ordinary cosmetics manufactured in
the country from June 201479
.
As consumer demand for safe and humane cosmetics increases
around the world, assessing the safety of cosmetics without using
animals is not only desirable, it is imperative. Governments must now
meet the global challenge to do the right thing for their citizens and
animals by ending animal testing for cosmetics.
Conclusion

“Alternatives
provide as much,
if not more, safety
for consumers.”

Cruelty Free International
16a Crane Grove
London
N7 8NN
United Kingdom
T: +44 (0) 20 7619 6995
F: +44 (0) 20 7700 0252
E: info@crueltyfreeinternational.org
www.crueltyfreeinternational.org

Meeting the Global Challenge - A Guide to Assessing the Safety of Cosmetics without Using Animals

Recommended

Recommended

More Related Content

Similar to Meeting the Global Challenge - A Guide to Assessing the Safety of Cosmetics without Using Animals

Similar to Meeting the Global Challenge - A Guide to Assessing the Safety of Cosmetics without Using Animals (19)

More from v2zq

More from v2zq (20)

Recently uploaded

Recently uploaded (20)

Meeting the Global Challenge - A Guide to Assessing the Safety of Cosmetics without Using Animals