SAFE USE OF BOTANICALSIN BEAUTY PRODUCTS

2

Safe use of botanicals in beauty products

FOREWORDAt P&G, product safety is paramount. Ensuring the safetyof our products for the people who use them is at the heartof what we do, and we have over one hundred in-house
experts who are devoted to the safety of all our products
and ingredients.
As the popularity of products with botanical ingredients
has increased, P&G has worked hard to develop a robust
safety approach which allows these complex mixturesof natural materials to be assessed and used. Contrary to
popular belief, ‘natural’ does not automatically mean ‘safe’.
Safe use of botanical ingredients is dependent onunderstanding the identity and composition of theirchemical components, the complex molecules andproteins that give rise to the product performance andexperience enjoyed by consumers worldwide.
However, understanding this complexity can bechallenging; plants can vary depending on local growing
conditions, botanical extracts can be prepared innumerous ways, and many botanical ingredients havenot been properly analysed and assessed against thestandards applied to other non-botanical ingredients, theirapplication based on hundreds of years of traditional use.
P&G’s safety scientists are passionate about theircontribution to the expert safety community. They havespent over a decade working collaboratively to address
the different challenges associated with ensuring thesafe use of botanical ingredients and have shared theirwork through numerous peer-reviewed publications
and presentations.
This white paper presents guidance for assessing the safeuse of botanicals in beauty products, with historical caseexamples, practical tools and background knowledge.
P&G hopes this work will help ensure all botanicals used
in beauty products will be safely enjoyed by consumers
throughout the world.
To find out more about P&G’s commitment to Responsible
Beauty, visit: https://us.pg.com/responsible-beauty/.

3

Safe use of botanicals in beauty products

INTRODUCTION
Botanical ingredients or extracts are naturally-occurring
complex chemical mixtures from plants (Roe et al.,2018). They can be sourced from the whole plant orfrom specific parts of the plant such as the leaves, roots,
flowers, fruits, seeds, or berries. Botanicals are typicallyused as extracts, oils or isolated by more complex
processes such as fermentation (Troyano et al., 2011).
Beauty products often include botanicals because of
their innate ability to moisturise, cleanse and perfume
or due to claimed qualities such as antioxidant, antiinflammatory and antimicrobial properties (Corazza etal., 2013).
A popular consumer belief is that natural ingredientsare intrinsically ‘safe’. However, there are many recorded
cases where beauty products containing botanicals
have caused serious adverse events such as irritation,
allergic contact dermatitis (inflammation and chronic
sensitivity of the skin), photosensitisation (skin reactions
to light), contact urticaria (localised swelling andredness of the skin) and difficulty breathing (respiratory
reactions) (Corazza et al., 2013; Kiken and Cohen,
2002). Often these adverse events are the result of
including botanicals without proper consideration for
identification, processing or exposure.
Between 2004 and 2010, a mail-order soap known
as ‘Cha no Shizuku’ was sold to 4.6 million customers
in Japan (Iwamoto et al., 2012). More than 2,100
individuals who used the soap went on to develop
allergic symptoms after ingesting natural wheat proteins(Yagami et al., 2017), with some cases of extreme
hypersensitivity and anaphylaxis. In these cases, thedevelopment of extreme Type I sensitivity observedin individuals were linked to the presence of large,hydrolysed wheat proteins used within the product(Burnett et al., 2018). Sales were swiftly discontinued in
May 2011 (Yagami et al., 2017).
Botanicals can be safely used in beauty products,
provided appropriate care is taken to ensure that thecorrect botanical is used, and enough toxicological
data are available to understand the risks and develop
adequate safety margins (Troyano et al., 2011). This is thesame standard applied to other ingredients used inbeauty products, including synthetics.
In this white paper, we describe the three steps thatP&G carry out to ensure the safe use of botanicals intheir beauty products. These include:

Identification of the plant,
the method of botanicalpreparation and extraction

Assessment of the daily dose
exposure to the botanical andsafety margin

Analysis of the related
botanical safety data,
including any adverse events
identified through post-market
surveillance and developmentof a robust safety margin4

Safe use of botanicals in beauty products

UNDERSTANDING BOTANICALS –IDENTITY, QUALITY AND PURITY
A robust understanding of botanical ingredients
will help select the right one for use in a beauty
product and help to minimise the risk of potential
adverse events. Natural biological variation, and thedifferent methods of processing, can impact theproperties of the desired botanical ingredient and its
subsequent functional role. Identifying, establishingand maintaining relationships with trusted botanicalsuppliers, is essential to ensuring high-quality,
reproducible botanical ingredients with consistentsafety profiles for use in beauty products.
Botanicals are rarely bought or sold in their raw
form; they tend to be supplied in waxes, oils oraqueous solutions, depending on how they havebeen prepared (Lesage-Meessen et al., 2015) and theirintended use. Due to these variables, it is importantto understand details about the plant from which
the botanical is derived as they may not be readily
apparent. A quality supplier will be able to provide
information about the plant genus and species oforigin, the part of the plant, and the method used toprepare the botanical ingredient. This information isparamount for a thorough safety assessment.

RBG Kew/ Jeff Eden

Ensuring quality and botanical fingerprinting
The need for quality suppliers is recognised
throughout industry, as it is possible for botanicalingredients to be contaminated or adulterated withcheaper materials. Many intentional or non-intentional
adulterants can have serious safety issues (Roberts etal., 2019). For example, grape seed extract, commonlyused as a dietary supplement due to its claimed
antioxidant effects, can be replaced or supplemented
by peanut skin or pine bark. These natural extracts all
contain different proanthocyanidins, but the productmay not provide the consumer with the intended
benefit without the specific type and amountof proanthocyanidin provided by grape seed extract
Some adulterants may be safe but fall short of theintended functional role of the botanical ingredient.
Consequently, purchasers need to be aware of thecommon adulterants and build relationships withquality suppliers to ensure the botanicals bought
are of known purity. Industry associations such as
the American Botanical Council keep purchasers
up to date on the most common adulterants andcirculating contaminant trends.

5

Safe use of botanicals in beauty products

Another way to ensure the quality of ingredientsprovided by a supplier is to test a sample of the
botanical for signature chemical markers – a botanical‘fingerprint’ (see aloe example in Figure 1). Thischemical fingerprint can be compared to knownreference standards using techniques such as nuclear
magnetic resonance spectrometry (1H-NMR), highperformance liquid chromatography with a UV
light detector (HPLC-UV), gas chromatography (GC)and thin-layer chromatography (TLC). GC, coupledwith time-of-flight mass spectrometry (ToFMS), hasbeen used to characterise the chemical signaturesof different types of tea, and benchmark markers ofquality for the tea production chain. TLC has beenused to verify the chemical signatures of someessential oils, and to evaluate the quality of thefinished product after development (Narasimhaji etal., 2019). The combination of GC and TLC have beenshown to reliably verify the contents of botanicalfingerprints in both a qualitative and quantitative
manner, allowing authentication of the botanicalingredient (Narasimhaji et al., 2019).
It is possible to use suppliers of United States
Pharmacopoeia-grade ingredients or International
Organization for Standardization (ISO) ingredients likeessential oils, which must fall within known ranges

Figure 1

Aloe vera –quality andbotanical
fingerprinting

for their perfume-like properties. Essential oils are
one example of a botanical material that have beenwell-characterised, with defined chemical constituent
ranges widely published.
Botanical fingerprinting can also be performed bybotanical experts such as at the Royal Botanic Gardens,
Kew, London. The Royal Botanic Gardens, Kew, has themost diverse botanical resource collection in the world,
including 7 million dried plants, 1.25 million dried fungi
and 50,000 plant DNA samples, as well as an extensive
seed bank. The Royal Botanic Gardens, Kew, can assist
with the selection of the best sources of sustainably
grown quality plants for optimum bioactivity. Theirscientists can also use a range of different analytic
methods - 1H-NMR, liquid chromatography-mass
spectrometry (LC-MS) and gas chromatography-mass
spectrometry (GC-MS) - to analyse samples against
their diverse plant collections, looking for comparable
botanical fingerprints as well as potential adulterants.
P&G works collaboratively with the Royal Botanic
Gardens, Kew, to identify plant extracts and ensure
the purity of botanical samples for certain botanicalextracts. P&G also completes rigorous and frequent
Good Manufacturing Practice Assessments (GMPA) on
its suppliers.

Aloe vera (L.) Burm. f. (also known as Aloe barbadensis Miller) is a succulent plant with a long
history of claimed health benefits. The gel within its leaves is widely used in beauty products for
its moisturising or hydrating effects to both skin and hair. Due to the worldwide demand for thisingredient, purchasers need to understand how the botanical composition can vary, in order tomonitor the quality of ingredients provided by suppliers so that their beauty product producesconsistent benefit for their customers.
Three main factors affect the botanical composition of aloe gel extracts; growing conditions, time
between harvesting and preparation, and method of processing. These factors affect the ratiosof chemical components, including sugars, enzymes, organic acids and polysaccharides, such asacemannan, the main bioactive ingredient (Marsh and Simmonds, 2020).
For beauty products, dry preparations of aloe are preferred for ease of transportation and productstability, but they can be adulterated with substances such as maltodextrin. In a study comparing
commercially available aloe powders from leading international suppliers, a multi-techniqueapproach was taken to determine the quality and authenticity of aloe powder versus fresh aloegel. 1H-NMR was used to identify the essential components of the aloe gel, which provide thecharacteristic signals that can be considered the ‘fingerprint’ of aloe. The same technique was also
used to distinguish between the expected aloe fingerprint and any potential adulterants, suchas maltodextrin. HPLC-UV provided information about the freshness of the product and the time
between the harvest and processing.
The study found the quality of the powdered samples to be inconsistent and, in some cases, very
poor. Some showed evidence of degradation and bacterial fermentation, and only three of the ninesamples analysed contained satisfactory amounts of the bioactive ingredient (Bozzi et al., 2006).

6

Safe use of botanicals in beauty products

DETERMINING CONSUMEREXPOSURE OF BOTANICALSFROM BEAUTY PRODUCTS
Beauty products are typically applied to the skin.
Exposure tends to be low as ingredients often remainon the skin and would have to be absorbed throughthe skin to enter the body; or enter the body throughan indirect and unintended route.

of use, but is important to note for products used on
the face, such as facial moisturiser and eye cream.
Ingestion is the final potential route of exposure andtends to relate more to food or dietary supplements.

Despite this low exposure, all ingredients, including
botanicals, have the potential to cause adverse effects
and so a quantitative toxicological risk assessment
is necessary to ensure product safety (Corazza et al.,2013). Understanding realistic human exposure to
botanicals through beauty products is vital fora proper evaluation of the ingredients used.

is essential in establishing the potential
level of human exposure. This includes how
much of a product is being used during
one application, the number of times in a
day the product is applied, the number of days it isapplied for, and whether it is a product to be usedchronically; which is often the case for most beauty
products. Consumers may also use multiple products
from the same brand, resulting in cumulative
exposure to the same ingredients over time.

Various regulatory agencies and authoritative bodies
may have standard methods to assess exposure(i.e. Europe’s Scientific Committee on Consumer
Safety [SCCS]). Best practice should be to consider
any appropriate regulatory agency approach andevaluate the following factors based on consumer
observation, diary studies, and other means to monitor
consumer exposure:

Exposure: Most beauty products are topicallyapplied, so the main routes of exposure
are dermal (rubbing in moisturiser to theskin or shampoo to the hair) or throughinhalation (breathing in deodorant sprays
or aerosols). Ocular (via the eyes) exposure can beconsidered accidental, as is not the intended area

Figure 2

Beautybotanical
ingredient A

Habits: Knowing how frequently a product is used

Duration: ‘Leave-on’ products such as moisturisers

will remain on the skin longer and sohuman exposure is higher than comparedwith ‘rinse-off’ products like shampoo.
However, despite ‘rinse-off’ products
seemingly staying on skin for a short time,
residual traces of the product can remain and be
found days after use.
By considering these different factors, the exposureof a botanical can be calculated and used for safety
analyses; Figure 2 (SCCS, 2012).

A beauty product contains 4% of the botanical ingredient A.
This ingredient contains 20% plant extract. The plant extract was made by adding 1 kg of plant to 9 kg
solvent via maceration. The following method was used to calculate the overall concentration of plantin the product:
Concentration in the product = Amount used in the product × concentration of the ingredientConcentration in the product = 4% × 10% × 20%
Concentration in the product = 0.08% (0.0008)
Consumer observation showed that 15 g of the product was used once per day. Only 1% of the
product was retained (not washed off).
To calculate the daily exposure to the botanical in the product, the following method was used:
Dermal exposure per day = concentration in the product × amount used (per day)×retention
Dermal exposure per day = 0.0008 × 15 g × 0.01
Dermal exposure per day = 0.00012 g or 120 µg

7Safe use of botanicals in beauty products

SAFETY ASSESSMENT
Once the botanical has been identified, checked, andthe daily exposure calculated, the next step of analysis
is the safety assessment. There are three parts to athorough safety assessment, and each outcome needs
to be acceptable for the botanical to be used in a
beauty product.
The three parts to a botanical safety assessment are:

Systemic safety: assessing
botanical safety inside thebody through three tiers ofevaluation (see Figure 3)

Skin safety: assessing thepotential for skin irritation,
delayed skin allergy (Type IV
allergy) and phototoxicity (skinreactions to light)

Immediate allergy safety:
assessing the potential for
immediate, food-type allergies(Type I allergy) such as hives,
asthma and anaphylaxis

Systemic safety
Tier 1 – is the botanical amount below a
threshold that is safe for exposure inside
the body?
The idea for a threshold under which exposure toa chemical would not result in any toxicity was first
proposed in 1967, and later developed in 1996 into
the Threshold of Toxicological Concern (TTC) (Munro
et al., 1996). TTC is a principle commonly used in foodsafety assessment which refers to the possibility of
establishing a human exposure threshold value for
ingredients below which there is no appreciable risk
to human health (More et al., 2019). Based on the ‘safe’doses for known substances in food, this corresponds
to a maximum dietary exposure of 0.15 μg per person
per day. (Mahony et al., 2020; Munro et al., 1996).
This is remarkably similar to the current challenge withbotanicals; potentially harmful chemical constituentsare known to be present in some plants, but not all

may have been tested for safety. A study was carriedout to apply this TTC approach to the concentrationof natural chemical constituents in botanicals. Basedon an extensive literature analysis, a maximum
TTC of 10 μg per person per day of botanical plantmaterial, based on dry weight, was determined to be
sufficiently protective (Mahony et al., 2020).
As discussed in the earlier example (see Figure2), a rinse-off product containing 4% of botanicalingredient A, corresponded to a total daily exposureof 120 μg per day. This value is over the TTC limit.
Determining whether a botanical ingredient is above
or below this threshold is known as a Tier 1 systemic
safety assessment. If the value is found to be above 10
μg per person per day, then subsequent Tier 2 or Tier 3
assessments are required.
To note, TTC can be used for botanical plants andsome extracts. TTC cannot be applied to extracts thatare selectively or wholly concentrated as this alters
natural ratios of chemical constituents found in theplant (Mahony et al., 2020). For example, it would not
be applicable to use TTC on a selectively concentrated
lavender extract that has been processed to containsignificantly higher levels of linalool, but where allother chemical constituents remain the same orat lower concentrations than what is found in thenaturally-occurring botanical. Further, it would not beapplicable to use TTC on a 200x concentrated aloevera extract in which all of the chemical constituentsare at a 200-fold higher concentration, but still in theirratios, than what is found in the naturally-occurring
botanical. Therefore, the value of 10 μg per person per
day should not be applied to concentrated botanicalmaterials like essential oils, or for botanicals witha known history of toxicity through preclinical or
clinical data.

8

Safe use of botanicals in beauty products

Figure 3:

Systemic safety assessment tiers for botanicalingredients in beauty products
TIER 1TIER 2TIER 3

Botanical TTC value

History of safe human use

Chemical constituent identification

Confirm botanical genus,
species, part of plant,
method of extraction

Conduct extensive literature
review (all reputable safety
data on the botanical)

Conduct chemical
constituent
identification (CCID)

Significant previous uses: food /traditional medicine

Calculate exposure to
the plant present in the
botanical ingredient

Separately identify andquantify each chemical
within the botanicaland assess eachchemicalExposure considerations:acute vs chronic

Over
TTC

Warnings and cautions:susceptible populations (e.g.
pregnant women)

Is the exposure < chemical TTC?
Are there sufficient data to support
exposure to each chemical present
in the botanical?

Any data / endpoint gaps
or concerns (genotoxicity,
developmental/
reproductive toxicity)
Botanical exposure< TTC (10μg/day)

No

Under
TTC

Sufficient data to establish SHU?

Yes

No

Yes

Botanical ingredients cannot be used
in beauty products if there are safetyconcerns related to special populations
and/or if the data only support acuteuse, even with significant evidence.

Conduct a literature review
looking for toxicology
data for each chemical(either naturally sourced orsynthetic)

Are there sufficient data
to support the exposure?

Yes

No

BOTANICAL CLEAREDFOR USE

Acronyms: Threshold of Toxicological
Concern (TTC), Safe human use (SHU)

Further examination
required using in silico
approaches

9

Safe use of botanicals in beauty products

Tier 2 – is there enough data on the botanical toconfirm safe human use?

Tier 3 – are the individual botanical chemical
constituents safe?

If the daily systemic exposure for the botanicalingredient is found to be higher than 10 μg per person
per day, then Tier 2 assessment is required. This step
involves scrutinising all reputable published data on
the botanical ingredient for known hazards, to showthat within reasonable and practical margins, it has ahistory of safe human use (SHU).

If the botanical exposure is above the TTC and ahistory of SHU data is not available, it is necessary
to quantify and assess the botanical at a chemical
constituent level (Baker and Regg, 2018). For chemical
constituent identification (CCID), the botanical isseparated into its constituent parts, and each of thesechemicals are separately assessed for safety using thechemical TTC or available toxicology data.

Data should be collected about the plant, different
methods of preparation, common usage, any safety
endpoint data (e.g. liver toxicity, genotoxicity or
carcinogenicity), epidemiology, and from current or
historical clinical studies, including surveillance reports
from adverse event databases such as the World
Health Organisation’s VigiAccessTM. If robust data
are found to support safe dietary use in humans orsafe chronic use as a traditional medicine, then the
botanical can be used in the beauty product (Galli etal., 2019).
However, if data are not found, or if safe chronic
human use is only available in a specific population,then the botanical cannot be used before it has beenfurther evaluated. As beauty products are available
for anyone to purchase, and the beauty market is notgoverned on a country-by-country basis, the use of abotanical in a beauty product should be proven safefor everyone, including sub-populations assumed tobe more sensitive – children, pregnant women andthe elderly.
If the botanical product does not have a provenhistory of SHU, then it is necessary to perform a Tier
3 assessment.

Figure 4

Ginkgo biloba
leaf – identifying
chemicalconstituents

Chemical constituent identification
Several established methods are available todetermine the constituents within a complex
mixture such as botanicals, including high- andultra-high-performance liquid chromatography (u/HPLC) (Guldiken et al., 2018), high-resolution mass
spectrometry (HRMS), ultraviolet (UV) detection, andcharged aerosol detection (CAD), Figure 4, (Baker andRegg, 2018). HPLC is a robust, yet versatile technique,commonly used to isolate constituents in natural
products through a series of mobile and stationary
phases (Sasidharan et al., 2011). HRMS can be used toquickly assign a molecular formula to constituents,
especially in the absence of reference standards,
which can be cross-referenced with online databases(Baker and Regg, 2018). Tandem mass spectrometry(MS/MS) studies are able to provide structuralclues to support conclusions by comparing against
fragmentation of known molecules in the same class,and aiding comparisons when using databases suchas mzCloud (Baker and Regg, 2018). UV detection
is a common technique for identifying unknown
chemicals, and is dependent on the presence of achromophore, a part of the chemical’s molecular
structure that absorbs UV light. This absorption canbe used to identify atomic structures and bondcharacteristics of specific chemicals. In components
without a chromophore, a CAD detector can beused (Baker and Regg, 2018). This method uses
polar charge, rather than light absorption, to identify
molecular components.

There are various methods to characterise complex botanical mixtures, but in the absence ofreference standards, it can be difficult to quantify individual chemical constituents.
Using Ginkgo biloba leaf as an example, Baker and Regg (2018) demonstrated an approachthat combines the different strengths of multiple instruments to allow the separation andidentification of complex botanical mixtures. Once separated by uHPLC, the components wereindividually identified and quantified by UV, CAD and HRMS, allowing for a direct correlation
between identity and relative ratio of different chemicals in the botanical mixture.
This technique may provide a platform for enabling in silico (computer modelling) of safetyassessments and providing quality assessments of botanicals without suitable reference standards.

10

Safe use of botanicals in beauty products

Once separated and identified, the chemical
constituents must be quantified for safety.

Some botanical ingredients, like essential oils used
for flavours and aromas, are well characterised, so it
may not be necessary to undertake CCID as publisheddata on its chemical constituents may be sufficient todecide on safety.

Final steps for systemic safety assessment
From the literature review in Tier 2, a safe dose range
for the botanical ingredient can be calculated. The
safety endpoints are ranked and the toxic effectthat occurs at the lowest dose, the ‘critical effect’ isidentified for the botanical. This will be the hazard
which will be used to calculate the safe range forthe entire botanical ingredient. To the ‘critical effect’dose, an additional ‘uncertainty factor’ safety margin
is added by reducing the safe dose level by at least
two orders of magnitude (SCCS, 2012). This is the
maximum botanical dose (see Figure 5).

While these techniques are very good at identifying
the chemical constituents in a botanical ingredientsample, they may not be able to isolate everything(Baker and Regg, 2018). For instance, during
chromatography, some of the complex mixture
can be lost due to volatility or through elution (itremains within the stationary phase and does notpass through to the detector). In addition, somechemical constituents may be present in such small
quantities that they are below the limit of detection(LOD) of the analytical method. If feasible, the LOD
for the analytical method is set at the chemical
TTC level. Although the resulting data may not
be a fully comprehensive representation of thecomplete botanical mixture, it is usually sufficient toestablish the main constituents in a sample. Throughthe optimisation of techniques used to separate
similar constituents in a complex mixture, and theability to present them to detectors separately for
identification, data interpretation and quantification
can be facilitated.

If data were insufficient for Tier 2 and a Tier 3 chemical
constituent identification was done, a safe dose range
will need to be calculated for each of the chemical
constituents identified based on the ‘critical effect’.
As in the Tier 2 botanical example, the process ofidentifying the maximum dose would be the same as
noted for the whole extract, but this time done at theconstituent level (SCCS, 2012).
Once the botanical and/or its chemical constituents
have completed the systemic safety assessment, thenext step is the site of contact assessment.

Figure 5:

How a safe range for a botanicalingredient is calculatedConcentration limit set by the
manufacturer

Safe range

00.1

0.1

Concentration limit for safety,
informed by research

Possibleharm

Safety margin

1

10

100

1000

10,000Concentration of botanical used in consumer products (gradient for illustrative purposes only)

All botanical ingredients in a beauty product are evaluated and confirmed to be well within the safe range for all
relevant toxicological endpoints

11

Safe use of botanicals in beauty products

Skin safety
Most beauty products are applied to the skin as the‘site of contact’. Site of contact analysis includes skinallergy (Type IV contact sensitisation), irritation andphototoxicity through dermal exposure (Troyano et al.,2011). As a rule, P&G does not formulate products withbotanicals that are known to cause irritation.
An important factor to consider is Type IV allergy ordelayed dermal contact sensitisation, similar to thatobserved by poison ivy. If an individual experiences
sensitisation or becomes allergic to a product oringredient, this usually persists for life. All ingredientsin a beauty product, including botanicals, areevaluated for contact sensitisation potential (seeFigure 6).

form, should be below this benchmark value. An
additional uncertainty factor is not added to thisbenchmark value as the natural extract will represent
a much smaller fraction of the whole botanicalcomponent (Troyano et al., 2011). The 10 μg per cm2
benchmark provides assurance of negligible contact
allergy risk and can also be used for dermal irritation,
photoallergy potential and phototoxicity (Troyano etal., 2011). Human Repeat Insult Patch (HRIPT) studies
should only be used for confirmatory purposes
once the botanical exposure relevant to the contact
sensitisation endpoint is determined to be safe.

The initial literature search on the botanical will
include any data relevant to contact sensitisation.
When available, these data will be used in the
Quantitative Risk Assessment (QRA) approach toevaluate safety for skin sensitisation (Kimber et al.,2017). When relevant data are not available for the
botanical, a maximum threshold value, based on
previous data, is needed to perform a skin sensitisation
risk assessment. A suggested benchmark for dermal
sensitisation is a dose of 10 μg per cm2. This value
was selected based on data from most potent skinsensitisers and is a protective value reflecting what
could be the ‘worst-case scenario’ (Troyano et al., 2011).
A botanical in its natural form, not in a concentrated

Figure 6

Essential oils
– the needfor safety
evaluation

Essential oils are widely used botanical ingredients, but at least 79 of them are known to cause
irritation leading to inflammation of the skin (de Groot and Schmidt, 2016).
In 2016, essential oil use was examined in adults at a Minnesota State Fair. Common reasons for using
essential oils included a desire for alternative treatments, a belief that essential oils were safer than
traditional therapies, or that they had turned to essential oils after the failure of standard therapies.
The survey also reported that in addition to physical conditions such as musculoskeletal, upper
respiratory and skin complaints, they were also being used for emotional reasons such as anxiety anddepression (Goodier et al., 2019).
Typical adverse events reported included rash, difficulty breathing, burning sensation, allergy anddiscomfort during urination after oral ingestion (Goodier et al., 2019). With the apparent rise inpopularity of essential oils, quality ingredients and appropriate safety limits are critical to prevent a
corresponding rise in contact allergic reactions

12

Safe use of botanicals in beauty products

IMMEDIATE ALLERGY SAFETY
Type I allergy is where the human body produces
IgE allergic antibodies in response to a foreign
substance (an ‘allergen’), leading to an inflammatory
response and the development of symptoms suchas conjunctivitis, hives, asthma, or anaphylaxis. Thiscan be triggered via four main routes of exposure toan allergen; inhalation, ocular, dermal and systemic(Krutz et al., 2019).
Allergens are often specific proteins, and each protein
can be assigned a level of allergenic potential. A
protein can be considered to have a low allergenic
potential if there is evidence of ongoing human
exposure without reports of an allergic reaction(Krutz et al., 2019). Databases such as Allergome canalso be used to understand whether the structure ofparticular proteins contains any IgE-binding antigens,the lack of which would provide good evidence
for the lack of allergenicity. Proteins with a higher
molecular weight (>2.5 kDa, or 20-30 amino acids)
should be considered potential allergens unless there
is significant evidence to the contrary.
A pragmatic approach to reducing this effect isavoiding known sources of allergens. If possible,
botanicals with a low protein content should be
used, unless the protein is the functional bioactive

component (Troyano et al., 2011). For example, P&G
generally avoid using globally recognised foodallergens, such as tree nuts, celery, and sesame seeds,in their beauty products (Troyano et al., 2011), as they
could elicit a response in individuals already sensitised(allergic) to these foods.
One of the most common routes of exposure by which
beauty products can cause Type I allergy is throughdirect inhalation of aerosols, such as deodorantand hair spray. It is also possible to indirectly inhale
more viscous topical products, such as shampoos,conditioners, and body washes, when small amounts
combine with fine water vapour in the shower. There
have also been cases where residual topical bodymoisturisers, applied to the body the day before, haveatomised off the skin in the shower the next morningand been inhaled (Kelling et al., 1998).
Botanical ingredients intended for use in direct spray
products and/or used (e.g. shampoo) or washed off
in the shower (e.g. body lotion) are tested for total
protein content and the resulting protein exposuremust be lower than the allergy benchmark before
they are included in a beauty product. Proposed
protein exposure should be below the protective
allergy benchmark of 0.1 ng/m3 (Troyano et al., 2011).

13

Safe use of botanicals in beauty products

Methods for quantifying
protein amount
Kjeldahl nitrogen analysis
Developed in 1883 by Johann Kjeldahl, this method
quantifies the amount of protein in a botanical bybreaking down the sample with strong acids andanalysing the amount of nitrogen released. This
process is suitable for botanical oils, waxes andaqueous solution samples and is internationallyrecognised (Neilson, 2019). The use of the Ion
Selective Electrode Detection system can increase
the sensitivity of the process to ~5ppm nitrogen. The
nitrogen results are multiplied by the factor 6.25
based on an AOAC compendia method to convert
the results into total protein (Troyano et al., 2011).
Many botanicals may contain other non-protein
nitrogenous chemical constituents and require atechnique with a greater degree of precision. The
Food and Agriculture Organization (FAO) of the United
Nations recently recommended the use of amino acidanalysis instead of the Kjeldahl method, but they also
noted that amino acid analysis often requires the useof sophisticated instrumentation, which may not bereadily available (Neilson, 2019).

Amino acid analysis
An amino acid analysis is used to quantify the aminoacid composition of an aqueous solution sample andestimates the molecular mass of a protein (Neilson,2019). First, the sample is hydrolysed under acidic andhigh-temperature conditions to break the proteins
down into amino acids, and then these amino acids
are separated using chromatographic techniques(Neilson, 2019; Troyano et al., 2011). Ion-exchange
chromatographic separation with post-column
ninhydrin derivatisation has been shown to provide
reliable data for the analysis of amino acids (Troyano
et al., 2011). Once the sample has been hydrolysed andseparated, each separate amino acid is detected andquantified. The estimated protein concentration of the
botanical sample can then be calculated:(total mass of recovered amino acids)(total mass of the sample)= total protein concentration(% weight)

Molecular weight distribution
Molecular weight distribution is used to analyseproteins in a botanical by relative mass. Sodium
dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) or size-exclusion chromatography (SEC)
can be used to analyse protein chains longer than
2.5 kDa (Neilson, 2019; Troyano et al., 2011). Aqueous

samples are usually analysed at three different
concentrations, the desired concentration (i.e. 0.1%),
ten times more concentrated (1%), and ten-folddiluted (0.01%). If the protein is not visible in either thedesired or diluted concentrations, it can be concluded
that the concentration of protein >2.5kDa in size is
acceptable (Troyano et al., 2011). SEC is an alternative
method of determining protein molecular weight
distribution, through the separation of molecules
based on their size using a column, where the largest
molecules are eluted first. Despite it having a lower
resolution than SDS-PAGE, SEC can be automated,and can also quantify based on comparison with the
UV absorbance of protein standards (Neilson, 2019).

Methods for characterisingprotein structure forpotential allergencity
Proteomics and bioinformatics
Recent approaches to the structural characterisationof proteins in a botanical include proteomics andbioinformatics analysis.
Proteomic analysis maps the specific protein
sequence. Databases such as NCBI and UniProt canthen be used to identify specific protein sequences
associated with allergenicity. Their 3D protein
structure can then be further analysed in orderto characterise structural or molecular features
relating to allergenicity (Maurer-Stroh et al., 2019).
The database AllerCatPro also stores information
about known allergens, and can be used to compare
and predict whether proteins are likely to have ahigh or low allergenic potential (Krutz et al., 2019)(see Figure 7).
Bioinformatics expand on this by collating large
amounts of existing data on known allergens (Krutz
et al., 2019). The protein sequence to be analysed canbe added to the database in the form of a ‘FASTA’
file, a text-based format for representing amino acidsequences. The database is then able to assess thesequence to identify any similarity to known allergens.
If there is a similarity, there is a high chance ofallergenicity. If there are no matches, the 3D structureof the protein is compared to that of other known
allergens. If similar structures are identified, thesequences of each are then compared to indicate thelikelihood of the sample being an allergen.
Once the three parts of the safety assessment haveconcluded and the botanical has been cleared, it canbe used in a beauty product.

14

Safe use of botanicals in beauty products

Figure 7:

Proteomic and Bioinformatic analysis:
assessing allergenic potential
Query protein (FASTA sequence)Protein is similar to
Gluten-like proteins

Yes

No

Gluten-like Q
repeats

Predicted 3D structureof protein is similar to 3D
structure of known allergens

Yes

Identity of aligned 3D
surface residues is >93%

No

Amino acid sequence is>35% identical with known
allergen over 80 residue window

Yes

No

Yes

No

3D surface
residues>93%

3D surface
residues<93%

80 residue
window>35%

Protein sequencehas 3 x 6-mers
identical to allergen

Strong evidence of allergenicity

Yes

No

3x 6-mers

No hits

Weak evidence of allergenicity
No evidence of allergenicity

15

Safe use of botanicals in beauty products

SUMMARY
The common belief that natural ingredients, suchas botanicals, are intrinsically ‘safe’ to use in beauty
products is false, as supported by the examples
presented in this white paper. However, botanicals
can be safely used in beauty products, provided
appropriate care and a range of analyses areundertaken including accurate identification of the
botanical, followed by defining consumer exposure,and a tiered safety assessment. This consumer
exposure and safety assessment follows the same
standards applied to man-made beauty ingredients.
Through these extensive analyses and safetyassessments, the risk of potential adverse events
can be minimised.

ensure product safety. One such example is theassessment of aggregate exposure needed for all
ingredients in a beauty product (Tozer et al., 2015;
Tozer et al., 2019). Another area of further study
may include the natural variations of chemical
constituents within a single species, and in different
parts of the same plant, depending upon themethod of processing.

Aside from the details discussed in this white paper,
further analyses may also need to be carried out to

For more information visit P&G’s Responsible Beauty
website https://us.pg.com/responsible-beauty/.

P&G strives to continually collaborate and share
information on the safe use of botanical ingredientsin beauty products with industry, manufacturers,
suppliers and retailers, based on over a decade ofresearch in this area.

16

Safe use of botanicals in beauty products

REFERENCES
Baker, T.R., Regg, B.T., 2018. A multi-detector chromatographic approach for characterisation and quantitation of botanical constituents to enable
in silico safety assessments. Analytical and Bioanalytical Chemistry 410, 11.
Bozzi, A., Perrin, C., Austin, S., Vera, F.A., 2006. Quality and authenticity of commercial aloe vera gel powders. Food Chemistry 103, 9.
Burnett, C., Bergfeld, W.F., Belsito, D.V., Hill, R.A., Klaassen, C.D., Liebler, D.C., Marks, J.G., Shank, R.C., Slaga, T.J., Snyder, P.W., Anderson, A.F., Heldreth,B., 2018. Safefty assessment of hydrolyzed wheat protein and hydrolyzed wheat gluten as used in cosmetics. Cosmetic Ingredient Review, 13.
Corazza, M., Borghi, A., Gallo, R., Schena, D., Pigatto, P., Lauriola, M.M., Guarneri, F., Stengeni, L., Vincenzi, C., Forti, C., Virgili, A., 2013. Topical botanicallyderived product: use, skin reactions, and usefulness of patch tests. A multicentre Italian study. Contact Dermatitis 70, 7.
de Groot, A.C., Schmidt, E., 2016. Tea tree oil: contct allergy and chemical composition. Contact Dermatitis 75, 14.
Galli, C.L., Walker, N.J., Oberlies, N.H., Roe, J.E., Edwards, J., Fitzpatrick, S., Griffiths, J.C., Hayes, A.W., Mahony, C., Marsman, D.S., O’Keeffe, L., 2019.
Development of a consensus approach for botanical safety evaluation - A roundtable report. Toxicology Letters 314, 7.
Goodier, M.C., Zhang, A.J., Boyd, A.H., Hylwa, S.A., Goldfarb, N.I., Warshaw, E.M., 2019. Use of essential oils: A general population study. Contact
Dermatitis 80.
Guldiken, B., Ozkan, G., Catalkaya, G., Ceylan, F.D., Yalcinkaya, I.E., Capanoglu, E., 2018. Phytochemicals of herbs and spices: Health versus
toxicological effects. Food and Chemical Toxicology 119, 12.
Iwamoto, S., Hagino, N., Tanimoto, T., Kusumi, E., 2012. Wheat-dependent exercise-induced anaphylaxis associated with a facial soap. Case Reports.
Kelling, C.K., Bartolo, R.G., Ertel, K.D., Smaith, L.A., Watson, D.D., Sarlo, K., 1998. Safety assessment of enzyme-containing personal cleansing
products: Exposure characterization and development of IgE antibody to enzymes after a 6-month use test. The Journal of Allergy and Clinical
Immunology 101, 9.
Kiken, D.A., Cohen, D.E., 2002. Contact dermatitis to Botanical Extracts. American Journal of Contact Dermatitis 13, 5.
Kimber, I., Gerberick, G.F., Basketter, D.A., 2017. Quantitative risk assessment for skin sensitization: Success or failure? Regulatory Toxicology andPharmacology 83, 5.
Krutz, N.L., Winget, J., Ryan, C.A., Wimalasena, R., Maurer-Stroh, S., Dearman, R.J., Kimber, I., Gerberick, G.F., 2019. Proteomic and bioinformatic
analyses for the identification of proteins with low allergenic potential for hazard assessment. Toxicological Sciences 170, 12.
Lesage-Meessen, L., Bou, M., Sigoillot, J., Faulds, C.B., Lomascolo, A., 2015. Essential oils and distilled straws of lavender and lavandin: a review ofcurrent use and potential application in white biotechnology Applied Microbiology ane Technology
Mahony, C., Bowtell, P., Huber, M., Kosemund, K., Pfuhler, S., Zhu, T., Barlow, S., McMillan, D.A., 2020. Threshold of toxicological concern (TTC) for
botanicals - concentration data analysis of potentially genotoxic constituents to substantiate and extend the TTC approach to botanicals. Food andChemical Toxicology.
Marsh, J.M., Simmonds, M.S.J., 2020. Aloe, activity, consistency, authenticiy and more: A review. Cosmetics and Toiletries 135, 9.
Maurer-Stroh, S., Krutz, N.L., Kem, P.S., Gunalan, V., Nguyen, M.N., Limviphuvadh, V., Eisenhaber, F., Gerberick, G.F., 2019. AllerCatPro - Prediction ofprotein allergenicity potential from the protein sequence. Structural Bioinformatics 35, 8.
More, S.J., Bampidis, V., Benford, D., Bragard, C., Halldorsson, T.I., Hernandez-Jerez, A.F., Bennekou, S.H., Koutsoumanis, K.P., Machera, K., Naegeli, H.,
Nielsen, S.S., Schlatter, J.R., Schrenk, D., Silano, V., Turck, D., Younes, M., Gundert-Remy, U., Kass, G.E.N., Kleiner, J., Rossi, A.M., Serafimova, R., Reilly, L.,
Wallace, H.M., 2019. Guidance on the use of the Threshold of Toxicological Concern approach in food safety assessment. EFSA Journal 17, 17.
Munro, I.C., Ford, R.A., Kennephol, E., Sprenger, J.G., 1996. Thresholds of toxicological concern based on structure-activity relationships. Drug
Metabolism Reviews 28, 9.
Narasimhaji, C.V., Meena, A.K., Maheshwari, B., Singh, A., Singh, R., Ilavarasam, R., 2019. Evaluation of an Ayurvedic polyherbal formulation throughTLC fingerprint profile of volatile oils: A quality contrl approach. Research Journal of Pharmacy and Technology 12, 6.
Neilson, S.S., 2019. Food Analysis, Fifth Edition ed. Springer, USA.
Roberts, G.K., Gardner, D., Foster, P.M., Howard, P.C., Lui, E., Walker, L., van Breemen, R.B., Auerbach, S.S., Rider, C., 2019. Finding the bad actor:
Challenges in identifying toxic constituents in botanical dietary supplements. Food and Chemical Toxicology 124, 8.
Roe, A.L., McMillan, D.A., Mahony, C., 2018. A tiered approach for the evaluation of the safety of botanicals used as dietry supplements: An industry
strategy. Clinical Pharmacology & Therapeutics 104, 12.
Sasidharan, S., Chen, Y., Saravanan, D., Sundram, K.M., Latha, L.Y., 2011. Extraction, isolation and characterization of bioactive compounds fromplants’ extracts. African Journal of Traditional, Complementary and Alternative Medicines 8, 10.
SCCS, 2012. The SCCS’s notes of guidance for the testing of cosmetic substances and their safety evaluation 8th revision. Scientific Committee on
Consumer Safety, 133.
Sica, V.P., Mahony, C., Baker, T.R., 2018. Multi-detector characterization of grape seed extract to enable in silico safety assessment. Frontiers in
Chemistry 6, 16.
Tozer, S.A., Kelly, S., O’Mahony, C., Daly, E.J., Nash, J.F., 2015. Aggregate exposure modelling of zinc pyrithione in rinse-off personal cleansing
products using a person-orientated approach with market share refinement. Food and Chemical Toxicology 83, 8.
Tozer, S.A., O’Mahony, C., Hannah, J., O’Brien, J., Kelly, S., Kosemund-Meynen, K., Alexander-White, C., 2019. Aggregate exposure modelling of vitamin
A from cosmetic products, diet and food supplements. Food and Chemical Toxicology 131, 16.
Troyano, E., Mcmillan, D., Sarlo, K., Li, L., Wimalasens, R., 2011. Approach to assesseing consumer safety of botanical ingredients with emphasis on Type I
allergy, in: N. Dayan, L. Kromidas (Eds.), Formulating, packaging and marketing of natural cosmetic products, First Edition. John Wiley & Sons, Inc.
Yagami, A., Aihara, M., Ikezawa, Z., Hide, M., Kishikawa, R., Morita, E., Chinuki, Y., Fukutomi, Y., Urisu, A., Fukushima, A., Itagaki, Y., 2017. Outbreak of
immediate-type hydrolyzed wheat protein allergy due to a facial soap in Japan. Journal of Allergy and Clinical Immunology 140, 3.