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Clear Stream project was inspired by a FlavourArt idea.
It is an in vitro study on living cells
to better understand electronic smoking effects
and its biochemical interaction with life.
To learn more about this topic I've interviewed:
Massimiliano Mancini, Chairman of FlavourArt
Prof. Anthony Milosz, Toxicologist of world renown
Elena Bocchietto, Biotechnology Specialist
Dr. Giorgio Romagna, Clear Stream project reviser.
Let’s start now our interview...
Mr. Mancini, how did Clear Stream project start?
In the last 2 years we noticed that
our flavourings were , and actually still are, bought
and used in a new way called "Vaping"
that is the use with electronic cigarettes.
Flavourings are usually mixed with food
and swallowed,
while in this case
they are mixed with a glycol or glycerine based liquid
and breathed with electronic cigarettes.
So we decided to forestall authorities’ requests
and study risks connected to this new kind of use.
A toxicologist and a doctor who uses e-cigarette
collaborate with us
and we are supported by external laboratories
specialized in cytotoxicity in vitro analysis.
Analysis process started with a formulations pro-active revision
and on toxicologist recommendation
we removed some compounds.
In general we removed some diketones
among which most common is diacetyl,
because it seems they are dangerous
if breathed for long periods.
The problem with diacetyl is that it’s an irritant it’s not...
it's not a carcinogen,
it’s irritates the airways
and it causes destruction of some of the finer bronchioles deep in the lungs.
Humans are very susceptible to this,
labs animals are more susceptible
to problems higher up in the respiratory tracks.
But it’s a problem in all species.
I get involved with FlavourArts when
I started doing some calculations
and found that
yes, it was plausible
that someone who’s using
large amounts of the buttery type flavour
would be exposing himself or herself
to concentration of diacetyl that are likely to produce
serious consequences.
FlavourArt as the leading flavour provider
you might say to the vape community,
was responsive to this issue
and started labelling compounds
that cannot be produced in a satisfactory manner
without using either diacetyl or one of the other compounds
which are for our practical purposes equivalent.
We started an in vitro acute toxicity evaluation program
at an acute level thanks to Abich laboratory
in order to identify all the possible risks
that may arise with these ways of use.
An vitro test is a test done on cellular culture of cells or
tissues of mammalian origin to generally evaluate
toxicity or also ingredients functionality or
completed products intended to be used on humans.
This is an alternative procedure to animal experimentations
that as you know, European Union is going to banish
in particular for non-pharmacologic products
such as cosmetic and topical products.
So, we are studying to develop some alternative procedures
able to give good projections of toxicity on humans.
Generally, we followed the UNI ISO 10993 standard
for medical devices that suggests
a list of standardized cells as 3T3 ones.
3T3 are skin fibroblast cells
and they are widely present in both
subcutaneous skin and several epitheliums.
Among epitheliums we find
all mucous membranes of breathing apparatus organs,
so they are the target cells of the product we study.
How does Clear Stream procedure work?
We simulate the use of e-cigarette,
so it is turned on ,
breathed by a pump in a glass becker.
The smoke is breathed for an established number of series
and it is then condensated in cells culture medium
and this soil is then used for cells testing.
Fibroblasts are in culture in a known number
up to 24/48 hours of exposure
with the substance under testing
in different dilutions and then we check
which is the concentration level
that causes mortality of 50% of cells.
Then we provide information about cellular mortality
at different concentration levels.
In this way it is possible to compare
different flavourings types and
check the cytotoxicity level in each flavouring,
thus identifying the most tolerated one.
We can estimate an acute toxicological risk of irritation
on human respiratory system caused by these flavourings
breathed by an e-cigarette.
Some laboratory results show that
cells viability is more than 100% with respect to reference value.
This could fall within test variability range because
cells are living organisms
and there is always some variability in tests.
The regulation accepts variability up to 15%,
thus it can be due to this.
Otherwise there could be also some substances
in a flavouring that help fibroblasts proliferation.
Food flavourings are accurately regulated at European level and
there is a list of what kind of substances could be used in flavourings.
This list is called
“Flavouring substances and natural sources of flavourings”.
It is obvious that we consider a different way of assimilation.
It is important to remind that
flavourings are all along added to tobaccos.
Generally they are burned and breathed,
while with e-cigarettes this does not happen
because the system is completely different
and we have basically the establishment of an aerosol.
We have a proactive approach to this topic,
we are not intended to gain popularity with our research.
Probably we will, but what we want is to be forward-looking
and to confirm our reliability as regards our proposal.
We do not want to earn easy cash
but to do a serious scientific study
with real data in order to assure our users’ health.
We believe e-cigarette will become a widespread
technology and will replace traditional cigarettes.
Moreover, if it is possible to make a leisure activity,
as smoking cigarettes, less dangerous
it is worth doing it.
What is your opinion about Clear Stream project?
Clear Stream project is a new approach to the
comprehension of toxicity factors due to electronic smoking.
Literature is full of gas-chromatographic analysis that
are useful to obtain some information about
chemical composition of e-cigarette vapour,
but we have indirect toxicological information,
because if we obtain a toxic substance as result of the analysis,
we can suppose the existence of a toxic phenomenon.
But, we cannot understand how it interacts with living structures.
With Clear Stream project we have this opportunity.
We can evaluate e-cigarette smoking behaviour
in biological contest.
What is the difference between acute toxicity and chronic one?
This study allows us to understand only instant toxicity phenomena
developed by the substance on cellular structures,
but we are not able to study long-term effects of
a prolonged and iterate exposure.
We wish to have this kind of information,
but it is not so easy to obtain them.
With respect to previous analysis of Clear Stream project
there is a remarkable change in results.
Unfortunately e-cigarette’s use is more complex than traditional one.
So it is necessary to have an expert user.
who deeply understands how it works
and that is able to use it
In addition, an expert user could understand some malfunctioning,
while a machinery does not recognize any abnormality.
In the first phase of our study we conducted tests
collecting samples by a device designed to deliver
a very small number of puffs with a filled cartridge,
but the number of puffs really done
was higher than the suggested one.
Then, the test probably generated an atomizer overheating
with plausible transformation or pyrolysis phenomena
on the residuals on the resistance.
and the production of substances
irritant or potentially toxic, not expected.
We want to pay attention on sensitization phenomena
that this kind of flavouring assimilation could cause,
since at a biological level of interaction with human organism
this kind of substance behaviour is well-known.
We have to keep in mind that flavourings
are made copying what nature did:
hence, compounds we use are normally present in foods.
Our body recognizes and it is able
to handle the substances that give flavour to a strawberry
whether they come from a strawberry or a flavouring.
On that basis, we can start to discuss toxicity issues.
We can say well, with cigarette for instance
it has taken hundreds of years to reach the understanding
that we have, by observation, by the laboratory studies,
understanding of how toxic cigarette smoke
is - tobacco smoke.
With e-cigarettes what is known about glycerine,
propylene glycol which are the two propellants if you will,
we know about the oral toxicity of this compounds
in animals,
we know how much it takes to kill an animal,
any substance would kill,
and we have some studies done on inhalation,
but these have not been very deep
or very complete studies,
so what is know right now for instance
about side effects of e-cigarette components
is not very much,
and this is generally true with most chemicals,
so this is not an unusual situation.
Officially, the picture is that both
propylene glycol and glycerine are low toxicity compounds,
both can produce disruptions in a system
if they are ingested in huge quantities, that’s true of water as well.
But in quantities that we’re exposed to as vapers,
chances are they don’t do very much, now will find out exactly later .
it is important to understand how much we know as a society,
as a species if you will, and then things we can observe and talk about,
but with the understanding that these are not official statements.
On FlavourArt website you can find a specific section
with study updates, laboratories results and much more.