Paper presented at IFEAT Dubai Conference September 25 – 29, 2016, “The Middle East: Challenges at the Historical Crossroads of the F&F Trade”

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WILL ALL THE FLOWERS BE GONE?

Kim Bleimann

Berje Inc. USA

kbleimann@berjeinc.com In 1966 some prescient industry leaders established “The Research Institute of Fragrance Materials” (RIFM). To give you a perspective of how long ago that was let me say that the infamous Environmental Protection Agency of the United States (EPA) was founded in 1970. I was 17 years old at the time. Our friends in Europe will recall that the present EU was founded in 1993, twenty-six years after RIFM.

Their expressed mission has been to assure, in conjunction with the International Fragrance Association (IFRA), the safety of the ingredients used by the Fragrance Industry. In symmetry, our flavour materials are looked after by the Flavor Extracts Manufacturers’ Association (FEMA), in the United States, with coordination with its EU counterpart, the European Flavour and Fragrance Association (EFFA), and its parent the International Organization of the

Flavor Industry (IOFI). These two independent groups, the flavour and fragrance folks, have expressed methodologies and focus based upon differing end-points of concern. Topical applications require protocols which are not the same as those for ingestion. The amount of money spent by RIFM over this time has been staggering: One can estimate that $66M have been spent for direct testing with an equal amount spent for scientific evaluation and assessment by the RIFM staff and the independent Expert Panel, the Expert Panel being the scientists and academics who verify the scientific validity and objectivity of RIFM safety assessments and research findings. It must be emphasised that the major portions of these funds have come primarily from the multi-national fragrance companies, not from us smaller corporations, or the essential oil houses. And, this is in addition to the huge amounts which they have spent annually testing their internal specialties. This, their data, is fed directly into the RIFM data base. Membership in RIFM is an expensive proposition, which we, Berje Inc. and 70 other members can assure you, it supports a current annual budget of $12.66M of which over $6.7M was spent on testing and research alone in 2015. For 2016 the budget is $9.02M for testing and research with increased efforts to add data to their base.

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Throughout their history of important work RIFM has been primarily focused upon individual chemical compounds. With their demanding and rigid standards they have published monographs and group summaries on 1,447 chemical substances with some 3,500 toxicological end-point evaluations. Defined standards have restricted usage for 187 materials and total bans have been issued on 84 specific compounds. Remember, this is our fragrance industry voluntarily restricting materials and removing them from our

perfumers’ pallets. This is a rare event for any industry in general and a laudable moral position which is often economically detrimental to many of our members. Our fragrance industry should be applauded for its intelligent forward thinking. However, the reasons for this historical single chemical approach to testing are not entirely clear. Either from oversight, neglect or because of the direction the folks who set the priorities for testing, Natural Complex Substances (NCSs) were not on their radar. And, in consequence, protocols and methodologies had not been established for NCS’ as they had for single substances. There had been an almost total neglect of essential oil testing per se. Following this unfortunate approach several of the chemical component compounds of our essential oils have had some worrying results. In 2011, as the incoming chair of The International Fragrance Association of North America (IFRA NA), I asked why this was so at what has become an infamous lunch, for me, with the two past IFRA NA Chairs, Dr Bill Troy, then with Firmenich, and Dr Sean Traynor.

As an aside, I am pleased to say that Dr Troy is a consultant who does currently work for IFEAT on our FEMA project; Dr Traynor retired from Takasago a couple of years ago. Lo and behold, he is an adventurer and a photographer of some note. At this lunch I was told categorically that the multi-nationals had assumed that we, the essential oil producers and dealers, were taking care of this testing. In disbelief I asked how it was that they had failed to consider how important essential oils were to their household and especially for their fragrance work. After all, where would fine fragrances be without Rose Oil and/or Jasmin Absolute? Or, Ivory Soap without Citronella Oil? Yikes. I was startled and quite angered that this entire swathe of our business had been so wilfully neglected. I must add, however, in their defence that yes, they were, and they still are, spending millions of dollars annually to assure the safety of their large volume products and their specialties. This dynamic needed to change. How would we ever “come up to speed?”

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A number of us essential oil people, led by the indefatigable Laure Moutet, some 15/20 years ago had insisted that oils should be considered as single substances. Why? Because they are, and they have an aesthetic, which supersedes the sum of their individual components. We believed that individual chemical components of oils should not be taken out of the natural context in which they exist. Basic biology would assume that plants produce these compounds for specific reasons such as predator control.

We knew that this component approach would get us into troubled waters if only because of the fact that many substances of our more complex oils, Vetiver for example, were unidentified, and are still not. Anyone familiar with REACH requirements and allergen identifications knows the depth and complexity of this problem, which directly results from this serious mistake ingrained into the EU thought process. The gravity of this situation became apparent six or seven years ago. Pending legislative initiatives, including REACH, brought into focus how sparse data truly was and how challenged the essential oil industry was by this neglect.

Yes, many components of the more common oils had been vetted, as they are integral to the perfumers’ pallet but some of this data we discovered was alarming and disquieting. That data showing that Methyl Eugenol, for instance, in isolation was a potential carcinogen is devastating. Look at the oils!

Rose Oil, Pimento Leaf and Berry Oils, Clove Bud Oil, Hyssop Oil, Basil Oil, Laurel Leaf Oil, Citronella Oil Sri Lanka, Citronella Oil Indonesian, Nutmeg Oil, Winter Savory Oil, Cinnamon Bark Oil, Cinnamon Leaf Oil, Melissa Oil, Cassia Oil, Aniseed Oil, Pinus Silvestris, Tea Tree Oil, Geranium Oil, Tarragon Oil, Tagette Oil, Valerian Root Oil, Calamus Oil, Tumeric Oil, Carrot Seed Oil, Caraway Oil, Eucalyptus Citriodora Oil, Eucalyptus Oil Globulus, Sage Oil Dalmatian, Camphor Oil, Howood Oil, Elemi Oil, Rosemary Oil, Lemongrass Oil, Wintergreen Oil, Black Pine Oil, Verbena Oil, Cardamom Oil, Mastic Oil, Cypress Oil. All of these essential oils contain some percentage of Methyl Eugenol. Can you see the gravity of this concern? If the European world view, which espouses the Precautionary Principle, holds sway all of these natural oils are in jeopardy for fragrance use. Fortunately we have had some encouraging news on Methyl Eugenol. What is neglected in this approach to science is that it fails to account for man’s relationship to his environment. Over tens of thousands of years of man’s evolution our bodies have learned to understand and assimilate this complex chemical fabric we inhabit. We have used, and we have consumed and we have lived with so many of these plants and spices since forever. To take them out of context and ignore the exposure levels has been misguided.

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Upon the initiative of your IFEAT Board and its Scientific Committee discussions produced a working document outlining an active exploration of the safety of NCS as entities not as an admixture of their components. In August/September 2013 this agreement was concluded with RIFM. IFEAT proposed to fund this work on essential oil safety for use in regulatory assessments. The fee was concluded at a rate of Sterling 100,000 per year for three

consecutive years for a total of Sterling 300,000, the first phase ending at the end of this year. The major points of this agreement state that in consideration of this funding, RIFM would warrant the following to IFEAT: 1. RIFM has taken the essential oils suggested by IFEAT as its priority. The first ones were those which were being scrutinised in our global socio-economic studies of essential oil production. We thought that this would dovetail nicely with those monographs.

2. RIFM will give us an annual accounting as to how these funds will be allocated and at the close of each year a summation of the work performed during that year.

3. IFEAT will be given credit, in the appropriate contexts, for having funded this work.

4. The results will be made available to those IFEAT related parties for illumination of REACH consortia.

5. IFEAT will be given access to the RIFM database for the essential oils which they requested be included on the priority list. Access for one or two members of the IFEAT Science Committee was agreed.

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I must commend the then IFEAT Chair, Ramon Bordas, as well as Alain Frix, Jens-Achim Protzen . . . and the ex-President of RIFM, Dr David Wilcox for their perseverance in getting this in place.

I am pleased to let you know that David is happily hiking and pursuing a passion for photography as well as Sean Traynor. He is really good - I would not have expected otherwise. So, this ends the history part of this talk.

Now, to the science, with which I am far less comfortable. I am indebted to Anne Marie Api for preparing this information for me, as I could not have done this myself. I will quote extensively from her so I give total credit.

So, what has RIFM been doing for us? To date IFEAT has selected 18 oils for evaluation with another 12 submitted this month. RIFM, upon receipt, has asked for compositional data from known industry users. During evaluation of the NCS of interest, the genotoxicity assessment can be conducted by using data on the NCS proper or by application of the “Threshold for Toxicological Concern” (TTC).

If this available data is shown to be lacking an initial genotoxicity evaluation is concluded by a BlueScreen©.

If the BlueScreen© assay is negative, meaning that it is not problematic, it would then go to the Reverse Bacterial Mutation test, that is the Ames test, OECD TE 471 and then to the in vitro micronuclear assays. An explanation of each of these tests below:

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1. BlueScreen© The BlueScreen© HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemicals compounds and mixtures. The aim of this project was to initially assess the genotoxicity and cytotoxicity of fragrance Natural Complex Substances in the BlueScreen© HC genotoxicity screening assay, using a protocol with and without metabolic activation. Results in the BlueScreen© HC assay are classified as either negative (N) or positive (P) with a corresponding LEC (Lowest Effective Concentration) for a positive result. The work was carried out at Gentronix Laboratories using a 96-well microplate format. The BlueScreen© HC uses the cultured TK6 cell strain (GLuc-T01), which are human lymphoblasts. Exposure to a genotoxic compound increases expression of GLuc, which is quantified at the assay endpoint by the detection of luminescence generated from the reaction of GLuc with a coelenterazine substrate added to the microplate wells just before measurement. Cell density is determined by the subsequent lysis of cells and addition of a fluorescent DNA addition of fluorescent DNA binding stain followed by assessment of the resulting fluorescence. Cytotoxicity as defined by lysis (the breaking down of cell membrane) which is measured by the addition of a fluorescent DNA binding stain followed by assessment of the resulting fluorescence. Fluorescence is proportional to cell proliferation, which is lowered by toxic analytes and luminescence intensity is proportional to the activity of the cell’s DNA repair system, which is increased by genotoxic analytes. Luminescence is normalized to the fluorescence signal to correct for variation in cell yield caused by cytotoxicity. It is important to note that BlueScreen© is not yet a fully accepted and validated methodology with results that must be reported before validation by some other method. For that reason it is a great screening toll from which we can tailor our subsequent testing. 2. Bacterial Reverse Mutation (Ames)

All studies were conducted in compliance with the US EPA Standards 40 CFR 792 (TSCA), the OECD Principles of Good Laboratory Practice (C(97)186/Final) and the Japanese Ministry of Health, Labor and Welfare Good Laboratory Practices (Ordinance Nos. 21 and 114, if applicable) in all material aspects with the following exception: Analyses to determine the concentration, uniformity and stability of the test substance dose formulations were not performed. Certificates of analyses and purity information were obtained from the sponsor. Each of the NCSs evaluated were tested in the Bacterial Reverse Mutation Assay (OECD TG 471) using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor induced rat liver S9. The assay was performed in two phases using the plate incorporation method. The first phase, the initial toxicity mutation assay, was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. Dimethyl sulfoxide (DMSO) or ethanol (EtOH) was selected as the solvent of choice based on the solubility of the test substand and compatibility with the target cells. 3. In Vitro Micronucleus Assay (OECD 487) Each NCS material was evaluated for cytogenicity in the in vitro mammalian cell micronucleus assay using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was conducted to establish the dose range for testing in the micronucleus test. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation. Dimethyl sulfoxide (DMSO) or ethanol (EtOH) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In solubility tests conducted, the test substances were soluble in either DMSO or EtOH and tested at the maximum concentration tested for solubility. Below a summary of the results on the eleven (11) NCSs tested to date. There are, at present seven (7) others in process.

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We are most pleased to report that all were negative for genotoxicity potential both with and without metabolic activation in the Blue Screen HC assay. Just recently we have been advised that Amyris, Cananga and Sclary Sage oil “passed” the Ames test. We await the results of the Ames on the other eight. All eleven of these oils are now under evaluation for the in vitro Micronucleus procedure. We are pleased and encouraged because several of these oils contain significant percentages of chemicals, which had netted unfavourable results when they had tested as a single chemical

substance. Something is tending to negate this negative aspect when they exist in their natural context. Methyl Eugenol, Citronellal, Linalool containing oils have had these encouraging BlueScreen© results. Rose Oil with approximately 2% Methyl Eugenol, Lavender and Sclary Sage contain significant percentages of Linalool and Linalyl Acetate. Eucalyptus Citriodora is +/- 75% Citronellal. Also encouraging are those favourable Ames test results on Amyris, Cananga and Sclary Sage. I hasten to emphasise caution, as these are very early days with only a small number of oils having been tested that is 11 out of some 250. This work, which RIFM is doing, truly supports all of our essential oil producers in a meaningful way. They are managing the testing and safety data requirements which are now mandatory in many parts of the world. Having clean scientific data supporting the safe use on our beloved oils will assist greatly in our advocacy with our respective governments. And, the marketing of oils will be constructively facilitated.

I have one worry which is that our EU brethren will invoke the Precautionary Principle and insist that Rose Oil, Lavender, and all of these other natural oils, should be dismissed from use because of its Methyl Eugenol content rather than consider its safety in its natural context. It is up to IFEAT to help dissuade this thinking. It should be our priority. Will all of our flowers be gone? Well certainly they could be, if we are not smart, diligent and scientifically rigorous.

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Kim Bleimann has worked in the flavour and fragrance industry since 1973 when

he joined Berje Inc. He assumed ownership and the presidency in 1981. Berje, Inc.

is a New York corporation, which has subsequently moved to Carteret, New Jersey

to a more modern and enhanced space. Berje has grown under his tutelage from a

company of nine employees to a firm which employs over one hundred people with

offices in Los Angeles, Cincinnati, Atlanta, and Plovdiv, Bulgaria where Berje is the

first ever American company to produce rose and lavender oils in Bulgaria. Berje,

now in its 66th year, has been an active and vital supplier of essential oils and

aromatic chemicals to the flavour and fragrance trades on a global basis. In early 2009 Berje

expanded their product offerings by forming an affiliation with The Whole Herb Company, a botanical

and spice supplier located in Sonoma, California. Professionally, Mr. Bleimann is a member of the

Flavor Extracts Manufacturers Association and the European Federation of Essential Oils. He has

served for three years, 2010-2013 as Chairman of the International Fragrance Association North

America which represents the interests of the fragrance manufacturers in the United States and an

independent adjunct to the International Fragrance Association which is the global association

representing the flavour and fragrance trades. In addition Mr. Bleimann serves as one of the Directors

of IFEAT where he chaired the Finance Committee from 2009 to 2015, and serves on the

educationally focused Study Tours Committee.