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This document contains copies of three recent literature reviews. Hyperlinks to each review are provided in the table below

Scope of review EU agency to Date Related which literature dossiers review sent A literature search on information relevant for EFSA August C/NL/09/01 the safety of GM carnation to humans, 2014 and including the safety of delphinidin and C/NL/09/02 potential allergenicity⃰. Updated assessment of the probability of gene Bureau GGO, NL October C/NL/13/02 dispersal from cut-flowers of the cultivated 2013 carnation (Dianthus caryophyllus) imported into Europe Updated review of potential toxicity and Bureau GGO, NL October C/NL/13/02 allergenicity of the acetolactate synthase (ALS) 2013 protein

⃰ Copies of the papers cited in this review are provided. Copies of literature review

An assessment of the probability of gene dispersal from cut-flowers of the cultivated carnation (Dianthus caryophyllus) imported into Europe

CONTENTS

Section Page 1. Baseline information; Dianthus biology 1 1.1 Taxonomy 2 1.2 Distribution 3 1.3 Reproductive biology 7 1.4 Inter- and intra-specific hybridisation 8 1.5 Weediness of Dianthus 10

2. Baseline information: carnation 11 2.1. Types of cultivated carnation 11 2.2. Carnation cultivation methods 12 2.3. Carnation utilization in Europe 13 2.4. Carnation import and distribution in Europe 13

3. Probability of gene dispersal from carnation 14 3.1. Introduction 14 3.2. Probability of gene dispersal by vegetative propagation 14 3.3. Probability of gene dispersal by seed set and seed distribution 14 3.4. Probability of gene dispersal by pollen distribution 15

4. Conclusions 16

5. Literature cited 18

1. Baseline information; Dianthus biology

Attachment B4 has been expanded from earlier versions included in previous marketing applications to the EU. As part of the regulatory process for the release of transgenic carnation in Australia, the Australian government has also produced an assessment of the biology and ecology of carnation (OGTR, 2006).

1.1 Taxonomy

Dianthus genus The Dianthus genus is a member of the Caryophyllacea, or pink, family and contains about 300 species. The genus is native to Europe, Asia, North Africa and the Arctic region, where one species is found (Hickey and King, 1981; Tutin and Walters, 1993). The second edition of Flora Europaea (Tutin, and Walters, 1993) lists 115 species, and 91 sub-species within 32 of these species. Seventy seven of the species listed are endemic to Europe.

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Recent studies have suggested that the Dianthus genus has one of the fastest rates of evolution in plants, possibly due to ployploidization (Weiss et al., 2002; Balao et al., 2009; Balao et al., 2011b). Dianthus chromosomes number ranges from 30 (diploid) to 105 (Weiss et al., 2002; Andersson- Kotto and Gairdner, 1931). Dianthus species are largely perennial, though some species, such as D. armeria, are annuals (Jurgens et al., 2002).

Dianthus caryophyllus In its unimproved, single flower form, Dianthus caryophyllus is called, in English, the clove pink or Grenadine (Britannica, 1999) or the clove gillyflower (Harvey, 1978). The English translation of the Latin word “caryophyllus” is “cloves” and caryophyllus was a commonly used name in the early taxonomy of the Dianthus genus (Smith, 1794). Clove pink was grown in the middle ages for its clove like perfume (Harvey, 1978) and was named by Linnaeus in Species Plantarum (De Langen et al., 1984). Dianthus caryophyllus may be taken as the type species for Dianthus.

The taxonomy of Dianthus caryophyllus has a long history and there are many sub-species and varieties of this species in the scientific literature. Dianthus siculus, D. arrosti, D. gasparrinii, D. virgatus, D. tarentinus and D. longicaulis were sometimes previously treated as subspecies or varieties of D. caryophyllus (Bacchetta et al., 2011). Bracchi and Romani (2010) include Dianthus caryophyllus var. inodorus L. and Dianthus caryophyllus var. orophilus (Jord.) Rouy & Foucaud in a recent checklist of Piacenza, Italy and Giardina et al. (2007) list a synonym of Dianthus gasparrinii as D. caryophyllus subsp. Gasparrinii. A worldwide Dianthus species, D. sylvestris, is known by the synonym D. caryophyllus ssp. sylvestris or var. sylvestris (Flora Piacentina, 2001; http://www.anthos.es). The e-flora of the French botany network (http://www.tela- botanica.org/site:accueil) lists nine varieties and eight sub-species of D. caryophyllus, in addition to the species itself. A compilation of the sub-species and varieties of Dianthus caryophyllus are shown in Table 1. The confusion in nomenclature can lead to mis-identification in the field – a problem recognized by Smith (1794) two centuries ago.

Table 1. Sub-species and varieties of Dianthus caryophyllus recorded in the taxonomic literature Dianthus caryophyllus subsp. sylvestris (Wulfen) Dianthus caryophyllus var. inodorus L., Rouy & Foucaud, 1896 ( = Dianthus sylvestris 1753 Wulfen ). Dianthus caryophyllus var. collivagus (Jord.) Cariot & Dianthus caryophyllus var. juratensis St.-Lag., 1889 Gren., 1865 Dianthus caryophyllus var. consimilis (Jord.) Rouy & Dianthus caryophyllus var. orophilus Foucaud, 1896 (Jord.) Rouy & Foucaud, 1896 Dianthus caryophyllus var. guyetanii (Jord.) Rouy & Dianthus caryophyllus var. saxicola Foucaud, 1896 (Jord.) Cariot & St.-Lag., 1889 Dianthus caryophyllus subsp. longicaulis (Ten.) Dianthus caryophyllus subsp. Arcang. godronianus (Jord.) Sennen, 1932 Dianthus caryophyllus var. longicaulis (Ten.) Dianthus caryophyllus subsp. virgineus P.Fourn., 1936 sensu auct. plur. Dianthus caryophyllus subsp. siculus (C.Presl) Dianthus caryophyllus subsp. Arcang., 1894 coronarius (Lam.) Bonnier, 1913 Dianthus caryophyllus var. coronarius L., 1753 Dianthus caryophyllus subsp. coronarius (Lam.) P.Fourn., 1936 Information;http://www.florealpes.com/fiche_oeilletsauvage.php?photonum=4&PHPSESSID=98ka413mmbrqb38otk1c bh8n5egkvi97# (Fleurs des Hautes-Alpes, de montagne, de Provence et d'ailleurs) and http://inpn.mnhn.fr/espece/cd_nom/133832/tab/rep (INPN, Inventaire National du Patrimoine Naturel, France.)

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The chromosome number of Dianthus caryophyllus samples collected from the wild has consistently been counted at 30 (Jones and Cooper, 1968; Gadella and Kliphuis, 1970; Hassall, 1978).

Carnation Carnations are double-flowered cultivars and in the general trade, botanical and horticultural literature carnation cultivars are considered to belong to the species Dianthus caryophyllus (Smith, 1794). The common name for Dianthus caryophyllus is carnation. However, the exact taxonomic and breeding history of carnation is not precisely known (Hughes, 1991; Harvey, 1978) and it is almost certain that carnation is a hybrid involving two or more Dianthus species, one of which is likely to be Dianthus caryophyllus (Hughes, 1991; Allwood, 1954). It is believed that carnation breeding began in the 1500’s in France (Holley and Baker, 1963). The double-flowered varieties of the carnation were known as an ornamental plant in Europe in the 15th century (Harvey, 1978). The herbarium specimen assessed by Linnaeus included a double-flowered carnation specimen (this may be viewed at http://www.linnean-online.org). More than one hundred years ago carnation breeding was well established in the USA, and today there are half a dozen large breeders in the world. The second edition of the International Dianthus register (1983) lists over 30,000 cultivars.

1.2 Distribution

Dianthus genus

The centre of biodiversity for Dianthus is southern Europe and the greatest range of Dianthus species are found in the south eastern European countries. Table 2 (adapted from Tutin and Walters, 1993) lists the number of species recorded in countries with the greatest diversity of Dianthus species. In Europe, most Dianthus species are found in the Balkan region and in the Mediterranean countries.

In North Europe Dianthus species are far less common, or even absent. For example, only six species are known in the British flora (Clapham et al., 1987), and five in Holland (Tutin and Walters, 1993). The majority of Dianthus species are not widely spread in Europe, and are confined to one or two countries, to specific mountain regions (Strid and Tan, 1997) or to alpine areas (Schwegler, 1979).

Table 2. European countries with the greatest diversity of Dianthus species Country Number of Dianthus species Former Yugoslavia 44 Bulgaria 39 Greece 37 Romania 32 Central + S.W. European Russia 31 Spain 26 Italy 24 Albania 21 France 20

There are six Dianthus species that have been found throughout the world. They are described below using information from Tutin and Walters (1993): • D. barbatus. Not a European species exclusively, D. barbatus is native to all Balkan countries and Eastern Europe. It has naturalized elsewhere after escape from cultivation. Copies of literature review

• D. armeria. The most widely distributed Dianthus species in Europe, as far north as Southern Sweden. This species has a world-wide distribution. • D. sylvestris. (Bacchetta et al., 2010). This species is highly polymorphic and closely related to D. caryophyllus. D. sylvestris is found in southern Europe and Mediterranean islands. There are six sub-species, three of these which are endemic to Europe, though the species itself is not. Dianthus sylvestris is found “from SE Spain to Greece and Northwards to the Swiss Jura and the Alps”. The typical habitat is rocky places (Polunin, 1980). • D. superbus. This species, which is not endemic to Europe, is found in all areas except much of the west and south of Europe (Tutin and Walters, 1993). There are three sub-species. • D. deltoides. This species is not endemic to, but is distributed in, most of Europe. It is rarer in the south (Tutin and Walters, 1993). Experiments have shown this species capable of establishment in restored meadows (Kalle et al., 2009). • D. carthusianorum. The only one of the six species which is possibly endemic, this species is found in south, central and western countries and has a very variable form.

In northern European countries the majority of Dianthus populations, if present, will be represented by one or more of the above six species (e.g. Perring and Walters, 1976; Van der Meijden, 1990). The distribution of these species in the North of Europe may be sporadic (Berten, 1990; Schonfelder and Ahlmer, 1990; Benkert et al., 1996). D. deltoides and D. carthusianorum are more common than the other four species listed above (Schonfelder and Ahlmer, 1990; Benkert et al., 1996).

Dianthus caryophyllus Unimproved Dianthus caryophyllus is reportedly only found wild in Mediterranean countries, and is rare. Whilst this area encompasses Spain, France, Greece, North Africa and Italy, Tutin and Walters (1993) state that the species is possibly only native to Sicily, Sardinia, mainland Italy and Greece. The Flora Europaea, accessed in September 2012 (http://193.62.154.38/FE/fe.html) still identifies France, mainland Spain, Italy (including Sicily and Sardinia) and Greece as countries where the species may occur wild. (Bracchi and Romani (2010) identified Dianthus caryophyllus in the province of Piacenza in Italy. Polunin and Huxley (1967) suggest the species occurs in France, Algeria and Morocco. In European floras the species is listed in Italy, Sicily and Sardinia (Zangheri, 1996), but not in floras of Greece (Strid, 1986; Strid and Tan, 1997; Turland et al., 1993), France (Guinochet and Vilmorin, 1973) and Andalucia (Valdes et al., 1987). In Mallorco Dianthus caryophyllus is only listed as a garden species (Barcelo, 1978). According to Ingwersen (1949) wild Dianthus caryophyllus can only be found commonly in specific coastal regions of Corsica. Figure 1 shows a photograph of Dianthus caryophyllus in the wild. This photograph is one of several recent collections of the species made in Southern France and posted on the website of the e-flora of the French botany network (http://www.tela-botanica.org/site:accueil).

The double-flower of cut-flower carnation varieties is now quite different to a flower from a wild Dianthus species, as shown in Figure 1.

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Figure 1. Comparison of the morphology of a flower from Dianthus caryophyllus (left hand side) to a cut-flower “standard” carnation variety (right hand side). The image on the left is extracted from http://www.isatis31.botagora.fr/en/eflore31/eflore31-v1.aspx (eFlore31, la flore en ligne de la Haute Garonne, France)

Ornamental Dianthus species The genus Dianthus contains several species which have been cultivated for hundreds of years for their ornamental value (Ingwerson, 1949). In the English language, these plants were called “pinks” from the 16th century, a term used to describe the whole Dianthus genus from Elizabethan times (Harvey, 1978). Table 3 lists some of the more commonly grown ‘pinks’ species. Photographs of most of these species are available on-line (Jagel, 2012).

Table 3. Dianthus species commonly grown for their ornamental value Species Common name Species Common name D. gratianopolitanus Cheddar pink D .plumarius Cottage pink, grass pink D.carthusianorum Carthusian pink D. alpinus Alpine pink D. superbus Fringed pink D. sylvestris Wood pink D. armeria Deptford pink D. chinensis Chinese pink, Rainbow pink, Indian pink, Japanese pink D. deltoides Maiden pink

Varieties of “carnation” have been reported very occasionally to have naturalized in the British Isles. These reports are summarized in Table 4. These accounts refer to garden plant varieties of pinks and carnation, which are not the same as cut-flower varieties and predate the development of modern cut-flower varieties by several centuries. Clement and Foster (1994) refer to carnation in the wild as "cultivars of this species (caryophyllus), native D. gratianapolitans and D. plumarius”.

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Table 4. Summary of reports of naturalization of Dianthus caryophyllus and hybrids in British floras Reference Dianthus species Comments by authors Maby, 1996 D. caryophyllus Probably introduced by Normans and naturalized in old walls in Rochester castle and Beaulieu abbey Perry and Ellis, D. caryophyllus “an escape of European origin” ( no further 1994 detail) Walters, 1993 D. caryophyllus X Single report from the year 1717 D. barbatus Stace, 1997 D. caryophyllus X Parentages of escaped garden pinks, some of D. gratianopolitans, which are occasionally naturalized on old walls. D. caryophyllus X D. gratianopolitans X D. plumarius Clement and D. caryophyllus Known for more than three centuries on the Foster, 1994 walls of Rochester castle. Clapham D. caryophyllus Occasionally naturalized on old garden walls. et al.,1987 Preston et al., D. caryophyllus “D. caryophyllus has been cultivated in Britain 2002 since the 16th century and is very common in gardens. It was first recorded in the wild in 1778, when it was discovered on the walls of Rochester Castle (E. Kent)”. ”Some historical records may be referable to hybrids with other species, which are also commonly grown.” “Not known with any certainty as a wild plant; recorded as doubtfully native in S. Europe.” D. caryophyllus X A single record in 1980. D. gratianopolitans D. caryophyllus X A tufted perennial herb which “occurs as a D. plumarius casual garden throw-out on rubbish tips and roadside verges”. D. caryophyllus X Parentages of escaped garden pinks, some of D. gratianopolitans, which are occasionally naturalized on old walls. D. caryophyllus X D. gratianopolitans X D. plumarius

Due to the international trade in ornamental plants, ornamental Dianthus species can now been found in many plants of the world, and on occasion have escaped from cultivation in Europe (section 1.5).

1.3 Reproductive biology

Dianthus genus The flowering period of wild Dianthus species is limited to summer in Europe (Table 5).

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Table 5. Flowering period for several Dianthus species in the wild. Alpine flowering periods are from Schwegler (1979) and Mediterranean data is from Strid and Tan (1997) Alpine Mediterranean Species Flowering months Species Flowering months armeria June - August armeria June - August arenarius June - September deltoides June - September barbatus June - September elegans May - July carthusianorum June - September giganteus June - August glacialis July - August gracialis June - August monspessulanus June – September integer July - August pavonius July superbus July - August sylvestris June - September sylvestris July - September

Pollination of Dianthus in nature is facilitated by insects (Knuth, 1908; Frankel and Galun, 1977; Erhardt, 1988; Jennersten, 1983 and 1984; Bloch et al., 2006) and is only effectively achieved by the Lepidoptera (butterflies, moths). Nocturnal hawk moths are one group of common insect pollinator (Balao et al., 2011a) and various Dianthus species are nocturnal- or diurnal-flowering, producing types and amounts of scents to attract specific types of pollinators (Jurgens et al., 2003, 2002; Jurgens, 2006). The chemical composition of several wild Dianthus species is described by Kishimoto et al. (2011). Lepidopteron insects are the only ones with proboscis long enough (up to 2.5 cm; Erhardt, 1990) to reach the nectaries, which are located right at the base of the flower in all Dianthus species (Hickey and King, 1981). Dianthus flowers are tubular (Figure 1), with strong bracts and calyx to exclude other insects (Knuth, 1908; Bloch and Erhardt, 2008). Table 6 lists pollinators of Dianthus observed in Europe.

Table 6. Reported Lepidoptera insect pollinators of Dianthus Dianthus species Moth genera Butterfly genera deltoides - Hesperia, Aphantopus, Aporia, Cyaniris, Ochlodes, Mesoacidalia, Polyommatus,Thymelicus superbus Macroglossum - carthusianorum Macroglossum Hesperia, Plusia, Inachis, Melanargia, Papilio, Thymelicus gratianopolitanus Macroglossum, Autographa, Papilio, Aglais, Cynthia, Pieris Hadena, Hemaris inoxianus Hyles chinensis - Plusia barbatus Macroglossum Pieris sylvestris Macroglossum, Hadena - glacialis Zygaena corymbosus Thymelicus, Aporia, Pieris monspessulanus Macroglossum - From Knuth (1908), Jennersten (1983, 1984), Erhardt (1988), Erhardt (1990), Erhardt and Jaggi (1995), Collin et al.(2002), Bloch et al. (2006), Bloch and Erhardt (2008), Yurtsever et al. (2010) and Balao et al. (2011a)

Dianthus species are largely protrandous, i.e. the anthers and pollen mature before the pistils (Knuth, 1908; Buell, 1952; Keane, 1989; Gargano et al., 2011). In some species gynomonoecious- gynodioecious mating systems also occur (Jurgens et al., 2002; Collin and Shykoff, 2003). Typically, when Dianthus flowers first open, the styles remain short and smooth. At this time the flower sheds pollen, and the styles are non receptive. As the flowers age the styles elongate and become covered on their inner surface with many hairs. If a flowers is not successfully pollinated Copies of literature review the styles continue to grow and curve. This can be seen in some cut carnation flowers, when the styles of some varieties protrude beyond the petals when they are left in the vase. If fertilization in Dianthus has been successful the flower collapses and the styles quickly shrivel. Though protrandy largely prevents self-pollination, D. deltoides and D.barbatus are known to readily self seed. D. armeria (Jurgens, 2006) and D. sylvestris (Jurgens et al., 2002; Collin and Shykoff, 2003) are also capable of selfing and the rare alpine species, D. glacialis is non-protrandous (Erhardt and Jaggi, 1995).

Jurgens et al. (2002) has provided information on pollen and ovule number in seven wild Dianthus species and supplemented this information with quantification of pollen grain size and style length in the same 7 species (Jurgens et al., 2012). Even within single anthers there are several populations of pollen grain size in Dianthus (Jurgens et al., 2012, Tejaswini, 2002). Bloch et al. (2006) established that in D.carthusianorum successful seed set was dependent on the number of pollen grains deposited on the stigma, with no seed set occurring with less than approximately 25 pollen grains.

Seed from Dianthus species is relatively short-lived. Mondoni et al. (2011) established the seed longevity in storage of Dianthus carthusianorum, D. sylvestris and D.glacialis to be three months or less. The same authors showed in comparisons of lowland and alpine populations of same species that alpine populations had shorter lived seeds. Seed from Dianthus rupicola had an optimal and rapid germination, at 15-25ºC (Lantieri et al., 2012). Seed of Dianthus morisianus was shown to be non-dormant and to exhibit non-light dependent germination. This species is unable to form a persistent seed bank, indicating the low long-term survivability of the seed (Cogoni et al., 2012).

Dianthus caryophyllus We are unaware of any scientific literature on the reproductive biology of wild Dianthus caryophyllus.

Viability of carnation pollen is varietal dependent. In varieties that produce low amounts of viable pollen, correct choice of anther stage for harvest is critical to optimize viability (Kim et al., 2005a). Tejaswini (2002) measured pollen grain size in 5 varieties of carnation establishing medium size grains were more likely to germinate. Though there was no significant difference between varieties there was a difference in mean germination by variety and pollen tube length was proportional to percentage germination and grain size.

Seed of Dianthus caryophyllus is light and 3 – 5 mm in length. Seed provided from a wild population collected in France is shown in Figure 2. This seed was collected in August 2009 and is partially viable; the seed was sown in August 2012 and approximately one-third of the seed germinated. As of April 2013 these plants were still in a vegetative state (Figure 2).

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Figure 2. Seed and seedling of Dianthus caryophyllus. Seed kindly provided by Jardin Botanique de Bordeaux, France. The red bar represents 5 mm. The lower plate shows a germinated seedling established as a young plant 6 months after germination.

Additional information on the reproductive biology of carnation is provided in section 1.4.

1.4 Inter- and intra-specific hybridization

In nature the Dianthus genus is characterized by a capacity for interspecific hybridization (Pax and Hoffmann, 1934; Ingwersen, 1949; Demmink, 1978; Castroviejo et al., 1990). Inter-specific hybridization has been utilized in breeding ornamental Dianthus species (Lee et al., 2005; Fu et al., 2011) and in fact inter-specific hybridization in Dianthus was one of the first scientific reports of plant hybridization, in the early 1700’s (Andersson-Kotto and Gairdner, 1931; Zirkle, 1934). D. knappii has been used as a genetic resource for yellow flower colour, D.superbus for its long feather like petals and D.barbatus for its multiple flower head. According to Tutin and Walters (1993) Copies of literature review natural hybridization in Dianthus is restricted to local regions where two species grow in high density and is particularly common in the Pyrenees. In these wild conditions there may be 4-5 flowering plants of any one species per square meter (Jennersten, 1984). The ploidy levels of inter- specific hybrids is not related to fecundity (Gatt et al., 1998). Carolin (1957) made 108 different interspecific crosses of Dianthus, and found 22% of crosses were fertile or sub-fertile, possibly due to embryo abortion (Buell, 1953). Bleeker et al. (2007) have stated Dianthus barbatus rarely hybridizes to the native D. deltiodes and D superbus in Germany. Andersson-Kotto and Gairdner (1931) have documented the compatibilities resulting from many inter-specific crosses within the Dianthus genus and indicate compatibility may not to be related to ploidy level.

Some carnation cultivars are self-sterile (Darwin, cited in Knuth, 1908 and Owens and Miller 2009 first observed this phenomenon in D. caryophyllus) and selfing, even under controlled conditions, produces either no seed or fewer viable seeds per capsule than cross-pollination (Mehlquist and Geissman, 1947; Zhou et al., 2013). Andersson-Kotto and Gairdner (1931) note that the self-fertility of carnation is markedly lower in double-flower varieties. Whilst there are some female sterile cultivars, (e.g. Copareve, Eolo) the cultivated carnation is not usually completely sterile (Silvy, 1978) and poor ‘selfer’ lines may produce seed after cross-pollination (Mehlquist and Geissman, 1947).

Interspecific hybridization with D. caryophyllus Efforts to hybridize Dianthus caryophyllus and other Dianthus species have been made to introduce useful horticultural genes into the cultivated carnation. Table 5 lists the species reported to hybridize to D. caryophyllus. Hybrids may be diploid, or triploid/tetraploid (Nimura et al., 2008). In crosses with D. caryophyllus Nimura et al. (2003) found that no seed set occurred when D. japonicus was used as the female. Wen et al. (1995) used embryo rescue to recover hybrids with D. chinensis and D.barbatus when D. caryophyllus was used as the female. As Table 7 shows, it has been possible to achieve a successful cross of D. caryophyllus to the widely spread European species D. barbatus, D. carthusianurom and D. sylvestris (this species is closely related to D. caryophyllus). All four of these species are diploid (2n = 30).

Table 7. Dianthus species reported to hybridize with D. caryophyllus Species Reference arenarius Holley and Baker, 1963; Umiel et al., 1987 barbatus Pax and Hoffman, 1934; Umiel et al., 1987; Wen et al., 1995 carthusianorum Demmink, 1978; Segers, 1987; Sparnaaij and Koehorst, 1990 chinensis Mehlquist, 1945; Demmink, 1978; Sparnaaij and Koehorst, 1990; Wen et al., 1995 deltoides Umiel et al., 1987 gallicus Holley and Baker, 1963; Andersson-Kotto and Gairdner,1931 giganteus Demmink, 1978; Sparnaaij and Koehorst, 1990 hungaricus Kishimoto et al., 2013 japonicus Nimura et al., 2003, 2008 knappii Holley and Baker, 1963; Segers, 1987; Sparnaaij and Koehorst, 1990 monspessulanus Holley and Baker, 1963 plumarius Gatt et al., 1998 sinensis Holley and Baker, 1963; Umiel et al., 1987 sylvestris Holley and Baker, 1963; Umiel et al., 1987; Demmink, 1987; Andersson- Kotto and Gairdner,1931 seguieri Holley and Baker, 1963 versicolor Sparnaaij and Koehorst, 1990

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Interspecific crosses using carnation can only be made in the glasshouse using manual intervention. Where carnation is the female parent this entails preliminary petal removal, manual pollination, calyx opening, final petal removal and fruit ripening on the plant (Sparnaaij and Beeger, 1973; Keane, 1989, Gatt et al., 1998).

The wild Dianthus species, Dianthus sylvestris, is found from South East Spain to Greece and Northwards to the Swiss Jura and the Alps. The typical habitat for this species is rocky places (Polunin, 1980). However, despite the fact carnation has been grown and traded in Europe on a large scale for decades there are no reports of the existence of hybrids between carnation and D. sylvestris in the wild in Europe (Tutin and Walters, 1993).

1.5 Weediness of Dianthus

In Europe, there have been escapes from cultivation of some Dianthus species (Tutin and Walters, 1993). These populations could be considered weeds because of their appearance in disturbed lands. However, they cannot be considered ecologically or economically important weeds (Holm et al., 1979; Guillerm and Maillet, 1982; Holzner and Immonen, 1982). Many Dianthus species are adapted to very specific geographical and climatic regions, such as alpine, rocky or sandy areas. This restricts their capacity for weediness.

As outlined, several Dianthus species are and have been grown for ornamental purposes for many centuries. Rare escapes from cultivation have been recorded in floras of Europe, such as for Dianthus barbatus (Bracchi and Romani, 2009). Clement and Foster (1994) describe a single population of Dianthus sylvestris in the UK, established on the rocks near Whitby harbour, in Yorkshire. Marco et al. (2010) established that cultivated Dianthus barbatus and Dianthus plumarius have not escaped in abandoned agricultural lands of Lauris villages, near Marseille, France. Schlaepfer et al. (2010) compared the seed germination characteristics of Dianthus armeria, considered to be a relatively invasive species, to D. carthusianorumnon. Dianthus armeria, in comparison to Dianthus carthusianorumnon germinated faster, produced more biomass and had a higher proportion of flowering plants.

Dianthus caryophyllus Dianthus caryophyllus is not a weed (Tutin and Walters, 1993). Specific references to the potential for escape from cultivation indicates very occasional survival, typically in rocky habitats like walls (Flora Piacentina, 2001; Dr César Delnatte, pers. comm.; National Botanic Garden of Belgium, pers. comm.; Societe Nationale des Sciences Naturelles et Mathematiques de Cherbourg, France. pers. com.). Brachhi and Romani (2009) have reviewed the alien species of the Emilia Romagna region of Italy and list Dianthus caryophyllus as an ornamental species that may have escaped cultivation and naturalized within population centres.

Carnation Despite decades of cultivation carnation has not become a weed, or escaped from cultivation, anywhere in the world. Each year over 10 billion flowers are produced for the world’s flower markets, and we have studied the floras of several areas with a significant area of cultivated carnation. There are no reports of naturalization of carnation in these floras: • Japan. Flora-Kanagawa, 1988; Ohwi, 1965. • Andean mountains, Ecuador. Jorgensen and Ulloa Ulloa, 1994. • Uplands of Kenya. Agrew, 1974. • Michoacan, Mexico. Garcia and Jimenez, 1993, Jimenez and Garduno, 1995. • Israel. Weissmann-Kollmann, 1965; Zohary, 1966. • Victoria and New South Wales, Australia. Harden, 1992; Willis, 1988 Copies of literature review

Cultivated clove pink (single flowered D. caryophyllus) is not winter hardy (MUS, 1996) as expected from the species natural distribution range on the coastal regions of the Mediterranean. The carnation will not survive outdoors in northern Europe. Florigene/Suntory has spoken to many carnation growers and has our own experience of the large scale production of transgenic carnation in Israel, Holland, Australia, Japan and Ecuador. Carnation has never been found growing wild, even in the immediate vicinity of carnation flower growing areas. In surveys carried out around the sites of production of carnation in Colombia, including composting areas, no carnation populations were identified (Chandler et al., 2008; section 2.2.1).

The cultivated carnation has no capacity to escape from cultivation as the crop possesses no vegetative propagation mechanisms and there are no opportunities for seed-set (section 3).

It could be possible, using tissue culture or other propagation techniques, to eventually deliberately cultivate plants from imported flowers.

2. Baseline information; carnation

2.1 Types of cultivated carnation

Cut flower varieties of carnation grow to 60 – 120 cm high, depending on variety, and produce flowers with a diameter up to 80 mm. Carnation can have from 30 – 100 petals per flower and the reproductive tissues of the flower are enclosed by the petals. In contrast, wild Dianthus species have an open flower, with the stigma and style easily accessible. The distinctive calyx of the genus Dianthus is seen in both flowers shown in Figure 1. The long tubular calyx is a morphological adaptation to pollination by moths and butterflies in the wild. In wild Dianthus species, including D.caryophyllus, the calyx ranges from 5 – 30 mm (Strid, 1986) and in cut flower carnation varieties from 25 - 40 mm. The calyx is relatively thick in the larger cut flower varieties. There are hundred’s of cut-flower varieties of cultivated carnation, categorized according to plant form, flower size and flower type. The two dominant groups, accounting for 96% of sales in the Dutch auctions, are standards and sprays, shown in Figure 3.

Figure 3. Spray type (left hand side) and standard type carnation (right hand side)

• Standards. These cultivars are grown under conditions in which a single large flower is produced per stem. Side shoots and buds are removed (a process called disbudding) to Copies of literature review

increase the size of the terminal flower. • Sprays (also called miniatures in the USA market). These cultivars are intended for cultivation to give a large number of smaller flowers (one per side shoot) per stem. Only the central flower is removed, allowing the laterals to form a 'fan' of stems.

2.2 Carnation cultivation methods

The cultivated carnation is vegetatively propagated and to produce plants for cut flower production cuttings are taken from vegetative 'mother plants' which are continually pruned to produce a high number of vegetative cuttings from axillary buds. These cuttings are rooted in conditions of high humidity, after treatment with auxin containing rooting powder, gels or solutions. Rooted plants may be planted in soil or grown hydroponically, and are kept for 1-2 years. Flowers are produced in flushes, beginning 3-5 months after rooted cuttings are planted. Picking of all flowers is essential and flowers must be harvested in tight bud (or closed bud for spray types) for distribution and marketing. The correct pick stage is strictly enforced by flowers entering the Dutch auction system, to ensure a satisfactory vase life in the hands of the consumer. A major problem for growers is the fungal wilt disease Fusarium oxysporum (OGTR, 2006). The occurrence of this fungus in untreated soil has led to relocations of growing areas in countries such as Spain, and adoption of cleaner cultural practice by the majority of European growers. Major pests of carnation are thrips, aphids and mites (OGTR, 2006).

Disposal of waste materials During carnation production, waste material is generated on a daily basis. This comprises vegetative material and flowers which are rejected due to quality problems, such as short length, or plants which have been uprooted when they reach the end of their productive life. In the latter case, irrigation is turned off to dry the plants before they are discarded. At both farms in South America where the transgenic carnation is grown, waste material is composted (Figure 4)

Figure 4. Composting areas in carnation production facilities in Colombia (left hand side) and Ecuador (right hand side)

As part of the regulatory compliance in Colombia, experiments have been carried out in which rooted carnation cuttings, uprooted cuttings and chopped plant material have been discarded for a period of up to 6 months in cleared plots and plots with existing vegetation. These experiments have shown carnation was unable to survive under these conditions. Copies of literature review

The vicinity of the compost heaps have also been inspected on an at least annual basis for the presence of established carnation plants. None have been found. In Colombia the plants growing near the compost heaps have been identified (Chandler et al., 2008). 100 species from 35 families were identified across 8 sample sites. The dominant family at farm locations was the Asteraceae, followed by the Poaceae. The species found at the most sites in the farm environments were Pennisetum clandestinum (kikuyu grass), Taraxacum officinale (dandelion) and Poa annua (annual bluegrass). No carnation (Dianthus caryophyllus) plants were found in any transect. The only Caryophyllaceae species found were Arenaria lanuginosa ( Michx.) Rohrb., Silene gallica L., Spergula arvensis L. and Stellaria media ( L.) Cirillo. Of these 4 species, only Stellaria media was identified at the composting area itself.

2.3 Carnation utilization in Europe

Historically, approximately 8 billion carnation flowers are consumed in Europe each year (Heinrichs and Siegmund, 1998). Approximately 20% of the European supply of cut carnation flowers is imported (largely into Holland, United Kingdom and Germany) from Colombia, Ecuador, Kenya, Israel, Morocco, Turkey and Zimbabwe (Heinrichs and Siegmund, 1998). Most of the flowers produced in Europe are grown in Italy and Spain. The data suggests that there have been in excess of 800 million carnation plants in cultivation on an annual basis in Europe for more than 30 years. On the basis of an average yield of 8 flowers this represents 6 to 8 billion flowers a year, the vast bulk of which would be consumed in Europe.

2.4 Carnation import and distribution in Europe

Information on the actual number of carnation flowers imported into Europe from outside the EU can be accessed from EUROSTATS. Table 8 provides an extract of the data, with details for Colombia and Ecuador. Carnations are imported into Europe by air freight, or by truck from Turkey and Morocco. Typically imports are handled by specialist importers, who provide distribution to wholesale and retail flower outlets. At the wholesale level these outlets could be wholesale florists or flower markets, such as Covent Garden in the UK. There are many specialist importers affiliated to the Dutch auctions. Wholesale florists and markets provide access to flowers for florists. Importers may also be, or may forward to, companies that specialize in provision of flowers for supermarket/ grocery/ garage chains. In these case final product (perhaps assembled in bouquets with other flowers), would be sleeved, labeled and distributed to individual stores. Flower longevity in this chain is summarized in Table 9.

Table 8. Number of carnation flowers imported into the Netherlands and the EU from 2009 - 2012. Units are million flowers Importing Exporting country country Year Colombia Ecuador All Countries Netherlands 2009 326 25 460 2010 313 19 439 2011 302 20 474 2012 326 17 592 All EU 27 2009 815 30 1,662 2010 780 26 1,587 2011 652 31 1,604 2012 631 28 1,593

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Table 9. The process chain for cut carnation flowers imported into Europe Step in process Duration (days) Maximum total duration (days) Harvest of flowers at farm 0 0 Processing, storage for flying 1- 14 14 Distribution to importers 1-3 17 Processing and distribution to arrival at final retail 1-3 20 destination Display at retail 5 25 Consumption “in the vase” –e.g. display and use by 3- 21 46 consumer before discarding

Until the time flowers are displayed for consumers, distributors maintain flowers dry in boxes, or in buckets with a small amount of water. During this time flowers are refrigerated, typically in large walk in cool rooms, and the flowers are closed, as the refrigeration prevents flowers opening.

In the consumers hands, at ambient temperature, and in water, flowers will hydrate, fully open and eventually senesce and die. Carnation vase life is determined by the age of the flowers from harvest, variety, and how well the flowers have been treated after harvest with preservative chemicals. Correct treatment prevents damage by ethylene, the compound that triggers senescence in carnation flowers which is produced 2 – 7 days after flowers are fully open. However, even treated carnation flowers will eventually dehydrate, senesce and die.

3. Probability of gene dispersal from carnation

3.1 Introduction

The dispersal of genes from an imported cut flower can be by three routes;

• Through vegetative spread of the discarded flower, leading to the formation of clonal populations. This possibility is discussed for carnation in Section 3.2. • Through the formation and dispersal of seed from the flower, as a result of self fertilization or fertilization with pollen from an external source. This possibility is discussed for carnation in Section 3.3. • Through the formation and dispersal of seed by a recipient plant, fertilized by pollen transferred from the flower. This possibility is discussed for carnation in Section 3.4.

3.2 Probability of gene dispersal by vegetative propagation

Carnation is vegetatively propagated (by cuttings) but the species does not spread vegetatively, i.e. the plant does not produce organs such as stolons, rhizomes, root-borne shoots, tubers, bulbs, corms or runners. Cuttings have to be struck in optimized conditions, and roots will not form on discarded untended materials, i.e. cut-flowers or old plants disposed of by growers or florists. This has been confirmed experimentally and by observation (see section 2.2.1).

3.3 Probability of gene dispersal by seed set and seed distribution

For gene flow by seed dispersal to occur in a carnation flower, the following events must all occur: • Arrival of viable pollen on the stigma. • Pollen germination and pollen tube growth to the ovule. • Fertilization. • Growth and maturation of the embryo and seed maturation on the cut flower. Copies of literature review

• Seed dispersal. • Seed germination and plant establishment.

Whilst fertilization of carnation, even with pollen from other Dianthus species, is theoretically possible, the probability of natural fertilization in carnation, even under cultivation conditions is extremely low. This is because of the physical barrier of the multiple petals. This barrier presents a significant obstacle to any potential pollinating insects that might cross pollinate within the flower growing area or be carrying pollen from external sources. During production flowers are picked closed, and exported to Europe at this stage.

Notwithstanding the fact that successful pollination is unlikely to occur, no seed set could occur on the cut flower. This is because the process of seed development takes from 5 weeks (Sparnaaij and Beeger, 1973; Gatt et al., 1998) to 2 months (Arthur, 1981). Cut flowers of carnation, even if treated with silver for increased vase-life cannot be kept in the vase for longer than 3 weeks. Separation of the flower from the plant would in any event deprive any developing embryos of essential hormones and nutrients, preventing maturation.

3 .4 Probability of gene dispersal by pollen distribution

For gene dispersal to be successful by pollen dispersal, viable pollen would have to be transmitted to a recipient plant and fertilization, seed set and seed dispersal occur. This sequence of events is extremely unlikely.

Standard and midi type cultivars of the cultivated carnation produce little or no pollen (Mehlquist et al., 1954; Kho and Baer, 1973; Nichols, 1976; Kim et al., 2005b) because in most commercial cultivars of this type anthers are converted to petals early in flower development (Arthur, 1981). Kim et al. (2005b) identified poor filament growth as another cause of poor pollen production. Spray type carnations produce more pollen than standards but amongst the spray types there are cultivar to cultivar differences in pollen production. In general, production of viable pollen by carnation is much lower than that of wild Dianthus species. It is known that water and nutrient stress can improve anther production in some standard cultivars and that temperature controls stamen and pollen production (Kho and Baer, 1973 Mehlquist et al., 1954). Carnations may be subject to high temperatures during the summer in southern Europe and this will reduce the potential for pollen production. Carnation pollen can be stored for one week (Sparnaaij and Beeger, 1973; Otten, 1991), placing a limit on the time available for transfer to a potential recipient. In enclosed environments such as glasshouses and greenhouses high humidity reduces pollen longevity.

The carnation flower opens out in the vase, increasing accessibility to the reproductive structures. In practice this is the only realistic opportunity for insect mediated pollen dispersal. However, at this stage of flower development any anthers are likely to have fallen from the stamens (Spaarnaaij and Beeger, 1973) and pollen would have significantly reduced viability, if any (Buell, 1952; Keane, 1989). If there were any viable pollen, it would still need to be dispersed to a suitable recipient plant.

As outlined in section 1.3, Dianthus is insect-pollinated and carnation pollen cannot be spread by wind. Any pollen produced is heavy and sticky (Jennersten, 1983) and buried deep in the flower. A survey of the atmosphere in the Netherlands, a country with a large carnation industry, failed to detect any carnation pollen (Driessen and Derksen, 1988). Only insects have the capacity to disperse carnation pollen, and only Lepidoptera could facilitate pollen transfer from carnation flowers to potential recipient plants. Common pests like thrips, aphids and spider mites are unlikely to move pollen and though ants can be found in flowers of all Copies of literature review types, ants are not likely to move much further than a few meters (Armstrong, 1979). Ants are also typical `nectar robbers' and their secretions usually kill pollen (Herrera et al., 1984; Gottsberger, 1989; Harriss and Beattie, 1991; Gomez and Zamora, 1992). Bees and wasps can be observed foraging in carnation flowers, but there is no evidence that this foraging may lead to cross pollination. As a precaution, bee movement is controlled in breeding houses, because petals are usually removed from flowers.

Assuming, theoretically, an insect were to access a carnation flower in a vase in someone’s home, and carry away any viable pollen that might be present, the probability of subsequently fertilizing a recipient, flowering, Dianthus plant is limited. There are several studies suggesting insect pollinators only move pollen hundred’s of meters from source when feeding (Price, 1984; Nilsson et al., 1992). In the extremely remote case that a seed was formed as a result of fertilization of carnation pollen onto a wild Dianthus species, there are limited environments where a plant could become established. In nature, Dianthus caryophyllus, like many Dianthus species is only found in suitable habitats, such as rocky walls, sand or limestone hills and outcrops. The seed of the putative hybridization event would probably have to germinate in such environments in order to become established in the wild. Many Dianthus species are found at high altitude on mountains (Strid, 1986) or on islands, not in carnation growing areas. These species are very unlikely to receive pollen from carnation plants. When wild Dianthus species are not in flower, which is for 8 – 11 months, there is no risk of gene dispersal from carnation as a result of fertilization and seed set in recipient plants.

Probability of intergeneric hybridization There are several species in the Caryophyllaceae which are widespread throughout Europe, and which are also considered serious weeds. These include Arenaria serpyllifolia, Polycarpon tetraphyllum, Sagina apetala, Stellaria media, Silene gallica, Silene vulgaris and Spergularia rubra. However, as far as we are aware, there are no reports of intergeneric hybridization in the Caryophyllaceae.

4. Conclusions

It is reasonable to conclude that the probability of unintentional gene dispersal from an imported carnation flower is nil because possible avenues for gene dispersal are not available.

There is no risk of gene dispersal by vegetative spread: Carnation does not spread vegetatively, i.e. the plant does not produce organs such as stolons, rhizomes, root-borne shoots, tubers, bulbs, corms or runners. Roots will not form on discarded or old cut-flowers. Florigene/Suntory has experience of large scale production of carnation in Australia, Japan, Colombia and Ecuador. Carnation has never been found growing wild, even in the immediate vicinity of carnation growing areas where waste material has been discarded or has been left for composting.

There is no realistic possibility that seed could form on an imported cut flower For gene dispersal by seed formation to occur from a cut carnation flower, the following events would all need to occur successfully; arrival of viable pollen on the stigma of the carnation, pollen germination, pollen tube growth to the ovule of the carnation, fertilization, seed formation and seed dispersal. Notwithstanding the fact that successful pollination of a carnation flower in a vase is highly unlikely, no seed set could occur. This is because the process of seed development takes at least 5 weeks on a plant – where the growth of any developing embryo could be sustained. A cut flower will remain in a consumers hand for three weeks at most before dying.

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There is no possibility that pollen could disperse from a cut flower and create a viable hybrid population There are several mutually exclusive facts that, in combination, indicate that potential pollen spread is not a feasible avenue for gene dispersal. Firstly, the potential for pollen spread from a cut flower is only theoretically possible; • In general, production of viable pollen by carnation is much lower than that of wild Dianthus species. • Hybridization of Dianthus in nature is facilitated by insect pollination and is only effectively achieved by the Lepidoptera (butterflies, moths). Pollen is not spread by wind. • The only point in the chain where insects could be reasonably expected to access flowers is when on display or in consumers hands. The physical barrier of the multiple petals presents a significant obstacle to any potential pollinating insects in less open flowers. • As a carnation flower opens out in the vase, any anthers are likely to have fallen from the stamens and any pollen would have significantly reduced viability. Secondly, were viable pollen to actually be produced and successfully dispersed by an insect vector, the realistic chance of a successful fertilization resulting in a wild hybrid population is also extremely unlikely. • Dianthus caryophyllus is very rare. • In commercial carnation production, flowers are removed. The high concentrations of carnation production in Europe are therefore not available as potential recipients. • While interspecific hybridization is know in Dianthus, hybridization is restricted to local regions where two species grow in high density and freely intermingle and is particularly common in the Pyrenees. In these wild conditions there may be 4-5 flowering plants of any one species per square meter. • The majority of Dianthus species are not widely spread in Europe, and are confined to one or two countries, to specific mountain regions or to alpine areas. • The flowering period of wild Dianthus species is limited to summer in Europe. When wild Dianthus species are not in flower, which is for 8 – 11 months, there is NIL risk of gene dispersal from carnation as a result of fertilization and seed set in recipient plants. • In the extremely remote case that a seed was formed as a result of fertilization of carnation pollen onto a wild Dianthus species, there are very limited environments where a plant could become established.

The long history of cultivation and utilization of carnation flowers in Europe supports the assertion that there is nil risk of gene dispersal. Carnation is not a weed in Europe. Despite hundreds of years of cultivation, and plantings in parks and gardens, it has not become a weed, or escaped from cultivation, anywhere in the world. Each year over 10 billion flowers are produced for the world’s flower markets. We have provided evidence for a lack of hybrids between cut flower carnation varieties and native Dianthus species in the wild in Europe, and for a lack of naturalized carnation in Europe. This is after decades in which very large numbers of carnation flowers have been imported into Europe. Large numbers of carnation are also grown in Europe. Compared to an imported cut flower, established plants of cut flowers varieties would theoretically be a more likely source material for gene dispersal than cut flowers. However, no hybrid between carnation and any other Dianthus species has ever been recorded in the wild

Expert opinion In earlier submissions for marketing approval of transgenic carnation imported into Europe we have provided two expert opinions. These expert opinions support the conclusions outlined above. Dr. Keith Hammett, an acknowledged expert on Dianthus breeding has concluded “that the likelihood of gene dispersal from cut flowers of fully double carnations to be highly improbable, if not inconceivable”

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Dr. Flavio Sapia stated; “The areas of the Coté d’Azur in France and of Riviera dei flora in Italy has been from 1920 to 1976 the biggest producer of carnation cut flowers in Europe. Even if this area has seen the growing of millions of plants and thousands of different varieties, never we had a cultivated carnation capable to survive in wild conditions” “for this reason the dispersal of a gene inserted in a carnation variety is, from my point of view, impossible..”

The Australian government (OGTR, 2006) concluded; “In Australia, gene transfer from carnations to any other plant species, even the most closely related naturalized Dianthus species, is unlikely due to the very low fertility of carnations.”

5. Literature cited

1. Agrew, A.D.Q. Upland Kenya wild flowers; a flora of the ferns and herbaceous flowering plants of upland Kenya. Oxford University press, United Kingdom, 1974. 2. Allwood, M.C. Carnations, pinks and all Dianthus. 4th edition. Allwood Bros Ltd. Sussex, UK, 1954. 3. Andersson-Kotto, I. and Gairdner, A.E. Interspecific crosses in the genus Dianthus. Genetica 13: 77 – 112, 1931. 4. Anon. Dianthus - a pert and sometimes fragrant border plant. http://www.jcsolutions.com/jcsgrower/peredian.htm, 12/01/99, 1999. 5. Armstrong, J.A. Biotic pollination mechanisms in the Australian flora - a review. New Zealand Journal of Botany 17: 467-508, 1979. 6. Arthur, A. Finding Fusarium tolerance - the easy part of carnation breeding. Grower, August 20 p.32, 1981. 7. Bacchetta, G., Brullo, S., Casti, M. and Pietro Giusso del Galdo, G. Taxonomic revision of the Dianthus sylvestris group (caryophyllaceae) in central–southern Italy, Sicily and Sardinia. Nordic journal of Botany 28: 137–173, 2010. 8. Balao, F., Casimiro-Soriguer, R., Talavera, M., Herrera, J. and Talavera, S. Distribution and diversity of cytotypes in Dianthus broteri as evidenced by genome size variations. Anns. Bot 104: 965 – 973, 2009. 9. Balao, F., Herrera, J., Talavera, S. and Dotteri, S. Spatial and temporal patterns of floral scent emission in Dianthus inoxianus and electroantennographic responses of its hawkmoth pollinator. Phytochemistry 72: 601–609, 2011a. 10. Balao, F., Herrera, J. and Talavera, S. Phenotypic consequences of polyploidy and genome size at the microevolutionary scale: a multivariate morphological approach. New Phytologist 192: 256-265, 2011b. 11. Barcelo, F.B. Flora de Mallorca, Volume II. Editorial Moll, Mallorca, 1978. 12. Benkert, D., Fukarek, F. and Korsch, H. Verbreitungsatlas der Farn- und Blutenpflanzen Ostdeutschlands, Gustav Fischer Verlag Jena, 1996 13. Berten, R. Limburgse plantenatlas 2, LIKONA, Limburgse Koepel Voor Natuurstudie, 1990. 14. Bleeker, W., Schmitz, U. and Ristow, M. Interspecific hybridisation between alien and native plant species in Germany and its consequences for native biodiversity Biol. Cons. 137: 248– 253, 2007. 15. Bloch, D. and Erhardt, A. Selection toward shorter flowers by butterflies whose proboscis are shorter than floral tubes. Ecology 89: 2453 – 2460, 2008. 16. Bloch, D., Werdenberg, N. and Erhardt, A. Pollination crisis in the butterfly-pollinated wild carnation Dianthus carthusianorum? New Phytol. 169: 699 – 706, 2006. Copies of literature review

17. Bracchi, G. and Romani, E. La flora alloctona della provincia di Piacenza (Emilia Romagna, Italia) e le sue variazioni dalla fine dell’ottocento a oggiparva Naturalia 2007-2009, 8: 169 – 231, 2009. 18. Bracchi, G. and Romani, E. Checklist aggiornata e commentata della flora vascolare della Provincia di Piacenza Società Piacentina di Scienze Naturali, Museo Civico di Storia Naturale di Piacenza, Piacenza, 2010. 19. Britannica. Encyclopedia Britannica Inc. 1999. 20. Buell, K.M. Developmental morphology in Dianthus I. Structure of the pistil and seed development. American Journal of Botany 39: 194-210, 1952. 21. Buell, K.M. Developmental morphology in Dianthus III. Seed failure following interspecific crosses. American Journal of Botany 40: 116-123, 1953. 22. Carolin, R.C. Cytological and hybridization studies in the genus Dianthus. New Phytologist 56: 81-97, 1957. 23. Castroviejo, S., Laniz, M., Lopez Gonzalez, G.,Montserrat, P., Munoz Garmendia, F., Paiva, J. and Villar, L. Flora Iberica; Plantes vasculares de la Peninsula Iberica e Islas Baleres, Volume II, Real Jardin Botanico, C.S.I.C., Madrid, 1990. 24. Chandler, S.F., Arunduaga, R.A., Luis, E., Lopez, J. and Claudia, A.P.O. 2008. The production of transgenic carnation and rose in Colombia; a survey for wild populations and related species. 10th International Symposium on the Biosafety of Genetically Modified Organisms (ISBGMO), Wellington, New Zealand. 25. Clapham, A.R., Tutin, T.G. and Moore, D.M. Flora of the British Isles. Cambridge University Press, Cambridge, 1987. 26. Clement, E.J. and Foster, M.C. Alien plants of the British Isle, Botanical Society of the British Isles, London, 1994. 27. Cogoni, D., Mattana, G., Fenu, G. and Bacchetta, G. From seed to seedling, a critical transitional stage for the Mediterranean psammophilous species Dianthus morisianus (Caryophyllaceae). Plant Biosystems 1: 1–8, 2012. 28. Collin, C.L. and Shykoff, J.A. Outcrossing rates in the gynomonoecious-gynodioecious species Dianthus sylvestris (Caryophllaceae). Am. J.Botany 90: 579 – 585, 2003. 29. Collin, C.L., Pennings, P.S., Rueffler, C. and Widmer, A. Natural enemies and sex: how seed predators and pathogens contribute to sex-differential reproductive success in a gynodioecious plant. Oecologia 131: 94 – 102, 2002. 30. De Langen, F.R., Oost, E.H. and Jarvis, C.E. Lectotypification of Dianthus caryophyllus L. and D.chinensis L. (Caryophyllaceae). Taxon 33:716 – 724, 1984. 31. Demmink, J.F. Interspecific crosses in carnation. In: Proceedings of the eucarpia meeting on carnation and gerbera. Alassio (Ed.Quagliotti and Baldi) p. 103-108, 1978. 32. Driessen, M.N.B.M. and Derksen, J.W.M. (1988). Pollenatlas van de Nederlandse Atmosfeer, Onkenhout, Hilversum, eerste druk. Cited in Otten, 1991. 33. Erhardt, A. Pollination and reproduction in Dianthus silvester Wulf. In: Sexual reproduction in higher plants. (Eds. Crest, M. et al) Springer-Verlag, Berlin. 502 pages, 1988. 34. Erhardt, A. Pollination of Dianthus gratianopolitanus. Pl. Syst. Evol. 170: 125 – 132, 1990. 35. Erhardt, A. and Jaggi, B. From pollination by Lepidoptera to selfing: the case of Dianthus glacialis (Caryopyyllaceae). Pl. Syst. Evol. 195: 67 – 76, 1995. 36. Flora-Kanagawa. Flora of Kanagawa, Flora-Kanagawa association, Kanagawa, 1988. 37. Flora Piacentina. Compendio del patrimonio floristico della provincia di Piacenza (Emilia - Romagna) Museo Civico Di Storia Naturale Di Piacenza Societa’ Piacentina Di Scienze Naturali, 2001. 38. Frankel, R. and Galun, E. Pollination mechanisms, reproduction and plant breeding. Monographs on theoretical and applied genetics. Springer-Verlag, Berlin, 281 pages, 1977. 39. Fu, X., Zhang, J., Li, F., Zhan, P. and Bao, M. Effects of genotype and stigma development stage on seed set following intra- and inter-specific hybridization of Dianthus spp. Sci. Hort. Copies of literature review

128: 490 – 498, 2011. 40. Gadella, T.W.J. and Kliphuis, E. Cytotaxonomic investigations in some angiosperms collected in the Valley of Aosta and in the National Park "Gran Paradiso" Caryologia 23: 363 – 379, 1970. 41. Garcia, C. and Jimenez, L. Estudio floristico de la cuenca del rio Chiquito de Morelia, Michoacan, Mexico. Flora del Bajio y de regiones Adyacentes, Fasiculo complementario IV, June 1993. 42. Gargano, D., Gullo, T. and Bernardo, I. Fitness drivers in the threatened Dianthus guliae janka (caryophyllaceae): disentangling effects of growth context, maternal influence and inbreeding depression. Plant biology 13: 96–103, 2011. 43. Gatt, M.K, Hammett, K.R.W, Markham, K.R and Murray B.G. Yellow pinks: interspecific hybridization between Dianthus plumarius and related species with yellow flowers. Scientia Hort. 77: 207-218, 1998. 44. Giardina, G., Raimondo, F.M. and Spadaro, V. A catalogue of plants growing in Sicily. Bocconea 20: 87 – 88. Herbarium Mediterraneum Panormitanum, 2007. 45. Gomez, J.M. and Zamora, R. Pollination by ants: consequences of the quantitative effects on a mutualistic system. Oecologia 91:410-418, 1992. 46. Gottsberger, G. 1. Floral ecology report on the years 1985(1984) to 1988. Progress in Botany 50:352-361, 1988. 47. Guillerm, J.L. and Maillet, J. Western Mediterranean countries of Europe. In Holzner, W and Numata, N (Eds). Biology and Ecology of Weeds, W. Junk, The Hague, 1982. 48. Guinochet, M. and Vilmorin, R. Flore de France, Vol 1. Centre National de la Rechere scientifique, Paris, 1973. 49. Harden, G.J. (Ed.) Flora of New South Wales. NSW University Press, NSW, 1992. 50. Harriss, F.C.L. and Beattie, A.J. Viability of pollen carried by Apis mellifera L., Trigona carbonaria Smith and Vespula germanica (F.) (Hymenoptera: Apidae, Vespidae). Journal of the Australian Entomlogy Society 30:45-47, 1991. 51. Harvey, J.H. Gilliflower and carnation. Garden History 6: 46 – 57, 1978. 52. Hassall, D.C. Taxon 27: 56 – 57, 1978. 53. Heinrichs, F. and Siegmund, I. Yearbook of the International Horticultural Statistics, Volume 46. Ornamental Horticultural Products. Institut fur gartenbauokonomie der universitat Hannover, 1998. 54. Herrera, C.M., Herrera, J. and Espadaler, X. Nectar thievery by ants from Southern Spanish insect-pollinated flowers. Insectes Sociaux, Paris. 31: 142-154, 1984. 55. Hickey, M. and King, C. 100 families of flowering plants. Cambridge University Press, 567 pages, 1981. 56. Holm, L., Pancho, J.V., Herbrger, J.P. and Plucknett, D.L. A geographical atlas of world weeds. John Wiley and Sons, New York, 1979. 57. Holley, W.D. and Baker, R. Carnation production: including the history, breeding, culture and marketing of carnations. W.C. Brown Co Ltd, Dubuque, Iowa, USA, 1963. 58. Holzner, W. and Immonen, R. Europe: An Overview. In: Holzner, W. and Numata, N. (Eds.). Biology and ecology of weeds. W. Junk, The Hague, 1982. 59. Hughes, S. Carnations and pinks. The complete guide. The Crowood Press, Marlborough, UK, 1991. 60. Ingwersen, W. The Dianthus. Collins, 128 pages, 1949. 61. Jagel, A. Dianthus deltoides – Hede-Nelke (Caryophyllaceae), blume des Jahres 2012. www.botanik-bochum.de/pflanzenportrats 2012. 62. Jennersten, O. Butterfly visitors as vectors of Ustilago violacea spores between Caryophyllaceous plants. Oikos 40:125-130, 1983. 63. Jennersten, O. Flower visitation and pollination efficiency of some north European butterflies. Oecologia 63:80-89, 1984. 64. Jimeniz, P. and Garduno, J.E. Listado floristico del estado de Michoacan seccion I. Flora Copies of literature review

del Bajio y de regiones Adyacentes, Fasiculo complementario VI, May, 1995. 65. Jones, K. and Cooper, S.S. Taxon 17: 420, 1968. 66. Jorgensen, P.M. and Ulloa Ulloa, C. Seed plants of the high Andes of Ecuador, 1994. 67. Jurgens, A. Comparative floral morphometrics in day-flowering, night-flowering and self- pollinated Caryophylloideae (Agrostemma, Dianthus, Saponaria, Silene and Vaccaria).Pl. System. Evol. 257: 233 – 250, 2006. 68. Jurgens, A., Witt, T. and Gottsberger, G. Pollen grain numbers, ovule numbers and pollen- ovule ratios in Caryophylloideae: correlation with breeding system, pollination, life form, style number, and sexual system. Sex Plant Reprod. 14: 279 - 289, 2002. 69. Jurgens, A., Witt, T. and Gottsberger, G. Flower scent composition in Dianthus and Saponaria species (Caryophyllaceae) and its relevance for pollination biology and taxonomy. Biochem. System. Ecol. 31: 345 – 357, 2003. 70. Jurgens, A., Witt, T. and Gottsberger, G. Pollen grain size variation in caryophylloideae: a mixed strategy for pollen deposition along styles with long stigmatic areas? Plant Syst. Evol. 298: 9 -24, 2012. 71. Kalle, H., Ari-Pekka, H., Pasi, R. and Juha, T. Seed introduction and gap creation facilitate restoration of meadow species richness. J. for nature conservation 17: 236 – 244, 2009. 72. Keane, A.T. Breeding new carnation cultivars. International Plant Propagation Society Combined Proceedings 39: 88-89, 1989. 73. Kho, Y.O. and Baer, J. The effect of temperature on pollen production in carnations. Euphytica 22:467-470, 1973. 74. Kim, Y.A., Joung, H.Y., Kwon, O.K. and Shin, H.K. Morphological observation of anther and pollen by stages of flower development for improvement of the breeding efficiency in carnation Desio. Kor. J. Hort. Sci. Technol. 23: 446 – 450, 2005a. 75. Kim, Y.A., Joung, H.Y. and Shin, H.K. Morphological observation of floral organs to investigate the cause of poor pollen formation in carnation Desio. Kor. J. Hort. Sci. Technol. 23: 440 - 445, 2005b. 76. Kishimoto, K., Nakayama, M., Yagi, M., Onozaki, T. and Oyama-Okubo, N. Evaluation of wild Dianthus species as genetic resources for fragrant carnation breeding based on their floral scent composition. J. Japan Soc. Hort. Sci. 80: 175 – 181, 2011. 77. Kishimoto, K.,Yagi, M., Onozaki, T., Yamaguchi, H., Hakayama, M. and Oyama-Okubo, N Analysis of scents emitted from flowers of interspecific hybrids between carnation and fragrant wild Dianthus species. J. Japan Soc. Hort. Sci. 82: 145 – 153, 2013 78. Knuth, G. Knuth's handbook of flower pollination, Vol, 2. Oxford University Press, 703 pages, 1908. 79. Lantieri, A., Salmeri, C., Guglielmo, A and Pavone, P. Seed germination in the Sicilian subspecies of Dianthus rupicola Biv. (Caryophyllaceae). Plant Biosystems 1: 1-4, 2012. 80. Lee, S.Y., Yae, B.W. and Kim, K.S. Segregation patterns of several morphological characters and RAPD markers in interspecific hybrids between Dianthus giganteus and D. carthusianorum. Sci. Hort. 105: 53 – 64, 2005. 81. Maby, R. Flora Brittannica. Sinclair-Stevens, 48p, 1996. 82. Marco, A., Lavergne, S., Dutoit T. and Bertaudiere-Montes, V. From the backyard to the backcountry: how ecological and biological traits explain the escape of garden plants into Mediterranean old fields. Biol. invasions 12:761–779, 2010. 83. Mehlquist, G.A.L. Inheritance in the carnation. V. Tetraploid carnations from interspecific hybridization. Proceedings of the American Society of Horticultural Science 46: 397-406, 1945. 84. Mehlquist, G.A.L. and Geissman, T.A. Inheritance in the carnation (Dianthus caryophyllus) III. Inheritance of flower colour. Ann. Mo. Bot. Gard. 34: 39-72, 1947. 85. Mehlquist, G.A.L., Ober, D. and Sagawa, Y. Somatic mutations in the carnation, Dianthus caryophyllus. Proceedings of the National Academy of Science USA 40: 432-436, 1954. 86. Mondoni, A., Probert, A.J., Rossi, G., Vegini, E. and Hay, F. Seeds of alpine plants are short Copies of literature review

lived: implications for long-term conservation. Annals of botany 107: 171–179, 2011. 87. MSU. Michigan state University extension plant database. 03, 01/01/96, 1996. 88. Nichols, R. Pollination, corolla senescence and sites of ethylene production. Glasshouse Crops Research Institute, Littlehampton. Annual Report, p.58-59, 1976. 89. Nilsson, L.A., Rabakonandrianina E. and Pettersson, B. Exact tracking of pollen transfer and mating in plants. Nature 360: 666-668, 1992. 90. Nimura, M., Kato, J., Mii, M. and Morioka, K. Unilateral compatibility and genotypic difference in crossability in interspecific hybridization between Dianthus caryophyllus L. and Dianthus japonicus Thunb. Theor. Appl. Genet. 106: 1164 – 1170, 2003. 91. Nimura, M., Kato, J., Mii, M. and Ohishi, K. Cross-compatibility and the polyploidy of progenies in reciprocal backcrosses between diploid carnation (Dianthus caryophyllus L.) and its amphidiploid with Dianthus japonicus Thunb. Sci. Hort. 115: 183 – 189, 2008. 92. OGTR. The biology and ecology of Dianthus caryophyllus L. (carnation). Office of the gene technology Regulator, Australia, November 2006. Available at www.ogtr.gov.au. 93. Ohwi, J. Flora of Japan, Smithsonian Institute, Washington DC., 1965. 94. Otten, A. Riscico-analyse voor een bloeiproef van transgene anjers (Dianthus caryophyllus) onder Pk2 - kasomstandigheden. Risk evaluation submitted to Dutch Government, 1991. 95. Owens, S.J. and Miller, R. Cross- and self-fertilization of plants – Darwin’s experiments and what we know now. Bot. Journal of the Linn. Soc. 161: 357 – 395, 2009. 96. Pax, F. and Hoffmann, K. Caryophyllaceae In: Die naturlichen Pflanzen Familien (Ed. Engler, A. and Prantl, K.) Volume 16c, p356-361. Duncleer and Humblot, Berlin, 1934. 97. Perring, F.H. and Walters, S.M. Atlas of the British Flora. E.P. Publishing Ltd., 1976. 98. Perry, A.R. and Ellis, R.G. The common ground of wild and cultivated plants. National museum of Wales, Cardiff, 166 pages, 1994. 99. Polunin, G. Flowers of Greece and the Balkans, Oxford University Press, United Kingdom, 1980. 100. Polunin, O. and Huxley, A. Fleurs du Bassin Mediterraneen, Fernand Nathan, Paris, 1967. 101. Preston, C.D., Pearman, D.A. and Dines, T.A. New Atlas of the British Isles. Oxford University Press, 910 pages, 2002. 102. Price, P. W. Insect ecology. John Wiley and Sons, New York, 1984. 103. Schlaepfer, D.R., Glattli, M., Fischer, M. and van kleunen, M.A. Multi-species experiment in their native range indicates pre-adaptation of invasive alien plant species. New Phytologist 185: 1087–1099, 2010. 104. Schonfelder, P. and Ahlmer, W. Verbreitungsatlas der Farn- und Blutenpflanzen Bayerns, Eugen Ulmer GmbH and co., 1990. 105. Schwegler, A. Bergflora: Bloemplanten van de Alpen en Scandinavische Landen – Dutch version dy M.A.Ijsseling and A. Scheygrond, Thieme, Zutphen, 1979. 106. Segers, T. The development of interspecific carnation hybrids. Acta. Hort. 216: 373- 375, 1987. 107. Silvy, A. Mutation breeding in carnation. In: Proceedings of the eucarpia meeting on carnation and gerbera, Alassio (Ed. Quagliotti and Baldi) p.91-102, 1978. 108. Smith, J.E. Remarks on the genus Dianthus. Transactions of the Linnaean Society of London 2: 292–304, 1794. 109. Sparnaaij, L D. and Koehorst-Van Putten, H.J.J. Selection for early flowering in progenies of interspecific crosses of ten species in the genus Dianthus. Euphytica 50: 211- 220, 1990. 110. Sparnaaij, L.D. and Beeger, G.W. The improvement of seed production for breeding purposes in the glasshouse carnation (Dianthus caryophyllus). Euphytica 22: 274-278, 1973. 111. Stace, C. New flora of the British Isles 2nd ed. Cambridge University Press, 1130 pages, 1997. 112. Strid, A. Mountain flora of Greece, Volume 1, Cambridge University Press, Copies of literature review

Cambridge, 1986. 113. Strid, A., and Tan, K. Flora Hellenica, Volume 1. Koeltz scientific books, Konigstein, Germany, 1997 114. Tejaswini. Variability of pollen grain features: a plant strategy to maximize reproductive fitness in two species of Dianthus? Sex Plant Reprod. 14: 347 – 353, 2002 115. Turland, N.J., Chilton, L. and Press, J.R. Flora of the Cretan area, HMSO, London, 1993. 116. Tutin, T.G. and Walters, S.M. Dianthus L. In: Tutin, T.G., Heywood, V.H., Burges, N.A., Chaler, A.O., Valentine, D.H., Edmondson, J.R., Moore, D.M., Walters, S.M., Webb, D.A. (Eds). Flora Europea, Second Edition, Vol 1. Cambridge University Press, 1993. 117. Umiel, N., Behjan, K. and Kagan, S. Genetic variation in carnation: colour patterns of petals, number of buds and arrangement of flower buds on the stems. Acta. Hort. 216: 355-358, 1987. 118. Van der Meijden, R. Heukels Flora van Nederland, Wolters-Noordhof, Groningen, 1990. 119. Valdes, B., Talavera, S. and Fernandez-Galiano, E. Flora vascular de Andalucia Occidental 1, Ketres Editoria SA, Barcelona, 1987. 120. Walter, M. Wild and garden plants. Harper Collins, 200 pages, 1993. 121. Weiss, H., Dobes, C., Schneeweiss, G.M. and Greimler, J. Occurrence of tetraploid and hexaploid cytotypes between and within populations in Dianthus sect. Plumaria (Caryophyllaceae). New Phytol. 156: 85 – 94, 2002. 122. Weissmann-Kollmann, F. A taxonomic study of Dianthus of Palestine and of neighbouring countries. Israel J. Bot. 44: 141 – 157, 1965. 123. Wen, G., Yu, J., Zhao, X., Fan, G., Lin, D. and Zhen, Y. Studies on carnation (Dianthus caryophyllus) species hybridization. Acta Hort. 405: 82 - 90, 2005. 124. Willis, J.H. A handbook to plants in Victoria. Melbourne University Press, Melbourne, Australia, 1988. 125. Yurtsever, S., Okyar, Z. and Guler, N. What colour of flowers do lepidoptera prefer for foraging? Biologia 65: 1049—1056, 2010. 126. Zangheri, P. Flora Italica. Cedam – casa editrice Dott. Antonio Milani, Padova, Italy, 1976. 127. Zhou, X., Gui, M., Zhao, D., Chen, M., Ju, S., Li, S., Lu, Z., Mo, X. and Wang, J. Study on reproductive barriers in 4x-2x crosses in Dianthus caryophyllus L. Euphytica 189: 471 – 483, 2013. 128. Zirkle, C. More records of plant hybridization before Koelreuter. J Hered. 25: 3-18, 1934. 129. Zohary, M. Dianthus L. In; Flora Palaestina Partone: Equisetaceae to Moring, 1966.

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Potential toxicity and allergenicity of the acetolactate synthase (ALS) protein

Contents

Section Page 1. Literature review 1.1 Nomenclature 2 1.2 Biochemical function of the ALS enzyme 2 1.3 Inhibition of ALS activity by herbicides 2 1.4 Characterization of ALS gene(s) 3 1.5 Isolation of ALS gene mutations conferring herbicide resistance 4 1.6 S4-Hra; ALS gene mutation in tobacco 5 1.7 Crops resistant to ALS-inhibiting herbicides 6 2. An assessment of the potential toxicity of the ALS protein from the ALS gene S4- Hra used in the transformation vector pCGP 1991 2.1 Plant toxins 7 2.2 The toxicity of tobacco 8 2.3 Exposure to carnation 8 2.4 Conclusions 8 3. An assessment of the potential allergenicity of the ALS protein from the ALS gene S4-Hra used in the transformation vector pCGP1991 3.1 Plant allergens 9 3.2 Allergenicity of tobacco 10 3.3 Allergens in flowers 11 3.4 The presence of contiguous amino acid sequences in S4-Hra homologous to 12 sequences in known allergens 3.5 Conclusions - Allergenicity of the S4-Hra mutant SuRB gene 13 4. Biosafety evaluation of food crops containing ALS gene mutations 4.1 Canadian assessments of imidazolinone-tolerant Clearfield™ crops 14 4.2 Food crops containing the SuRB gene mutation S4-Hra 15 4.3 Transgenic flax 15 4.4 Animal feeding studies 15 5. Overall conclusions 17 6. Literature cited 17

1. Literature review

1.1 Nomenclature

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Acetohydroxyacid synthase (EC 4.1.3.18) is an enzyme with two metabolic roles (Duggleby and Pang, 2000; Nelson and Duxbury, 2008). The anabolic role is in the early steps of branched chain amino acid biosynthesis. The catabolic role, confined to certain micro-organisms only, is in the formation of butanediol. In the plant literature, acetohydroxyacid synthase is very often referred to as acetolactate synthase. For ease of reference to cited literature, acetolactate synthase is the name used for the enzyme in this document. Throughout this text the abbreviated name of the enzyme and gene is used (ALS).

1.2 Biochemical function of the ALS enzyme

The reviews by Duggleby and Pang (2000) and Duggleby et al. (2008) provide an overview of ALS function in micro-organisms and plants. • Acetolactate synthase is found in all plants, in bacteria (Nelson and Duxbury, 2008) and in fungi. Animals do not synthesize the branch chain amino acids. • In plants, ALS catalyses the biosynthesis of branch chain amino acids leucine, valine and ioleucine through the reactions shown in the figure below (extracted from Singh and Sander

(1995);

• Co-factors required for ALS activity are thiamin diphosphate (ThDP), metal ions (generally 0.1 to 10mM Mg2+) and flavin (FAD). • ALS activity is subject to feedback inhibition (Chen e al., 2010). Valine, leucine and isoleucine are all feedback inhibitors, but to different degrees in different species. ALS enzyme is very labile, and is difficult to purify, as it occurs at low concentrations. Southan and Copeland (1996) have purified and characterized the enzyme in wheat but carnation ALS has not been isolated.

1.3 Inhibition of ALS activity by herbicides

The ALS enzyme is inhibited by four classes of herbicides. The inhibition caused by herbicides can be reversed by provision of valine and isoleucine to pea cultures, restoring the ALS function (Ray, 1984). ALS-inhibiting herbicides are the sulfonylureas (Chaleff and Ray, 1984; Ray, 1984), imidazolinines (Shaner et al., 1984), triazolopyrimidines (Subramanian et al., 1990) and pyrimidines. The mechanism of inhibition (Duggleby et al., 2008) is through the binding of these Copies of literature review herbicides to a relic quinone-binding site in the ALS enzyme (Schloss et al., 1988), rather than to the substrate binding site. ALS-inhibiting herbicides have no structural similarities to the substrates (pyruvate and α-ketobutyrate) or cofactors (thiamine pyrophosphate, FAD and magnesium) necessary for ALS function (Schloss et al., 1988).

1.4 Characterization of ALS gene(s)

In plants, ALS is nuclear encoded but chloroplast localized (Mazur et al., 1987). Duggleby and Pang (2000) indicate that in all species at least one ALS gene is expressed constitutively. Depending on species there may be between 1 and 7 copies of the ALS gene (White et al., 2003). Sugar beet and Arabidopsis only have one gene (Mazur et al., 1987; Keeler et al., 1993). Ouellet et al. (1992) investigated the ALS gene in Brassica napus. Five genes were identified, which were expressed constitutively in some cases, or in a tissue specific fashion in other cases. Cotton has six ALS genes. Fang et al. (1992) showed maize has two ALS genes, which in common with all ALS genes studied in plants has no introns.

Conservation Within a species the ALS genes are highly conserved – the two genes in maize are 94% identical in the coding region, for example (Fang et al., 1992). Between species there is also a high degree of conservation. Based on aligned amino acid sequences, two of the genes in cotton, which were constitutively expressed, were co-linear to the genes from both Brassica napus and tobacco (Grula et al., 1995). Le et al. (2003) has shown that seven methionine residues (numbered 332, 347, 350, 489, 512 and 569 in tobacco) were conserved in all ALS sequences they examined.

Tobacco ALS Mazur et al. (1987) first isolated the ALS gene, from Arabidposis and tobacco. In tobacco there are two unlinked ALS genes, SuRA and SuRB (Chaleef and Mauvais, 1984; Chaleef and Bascomb, 1987). The tobacco ALS gene has been studied in great deal, primarily through site-directed mutagenesis. This research has found amino acid residues 141, 219,372, 376 and 512 and 652 are located at the active site, and mutations inactivate the enzyme and prevents binding of the cofactor FAD (Chong and Choi, 2000; Yoon et al., 2002; Le et al., 2005).Chong and Choi (2000) determined that amino acid pro187 was essential for maintaining the resistance of the enzyme to fragmentation and oxidation. Using site-directed mutagenesis Yoon et al., (2002) identified a lysine residue (position 255) which was substituted in mutations showing broad herbicide resistance, and so likely to be in a domain at which all herbicides bind.

ALS gene expression in tobacco has been studied by Keeler et al. (1993). SuRA and SuRB are both expressed in tissues with the highest metabolic activity (presumably due to the need for amino acids for protein biosynthesis), with a 3 to 4 range in expression in different tissues. The SuRB gene is expressed at higher levels than the SuRA gene. The SuRA and SuRB genes are highly conserved, with a predicted amino acid divergence of 0.7% (Lee et al., 1988).

1.5 Isolation of ALS gene mutations conferring herbicide resistance

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Herbicide resistant ALS gene-mutants have been isolated in many plants. Examples are shown in Table 1.

Table 1. Mutations of the ALS gene conferring herbicide resistance Mutation Characteristic of mutant Species/citation Single amino acid Multiple herbicide resistance. Feedback Brassica napus (557 Trp to Leu) inhibition unaffected. (Hattori et al.,1995) Single amino acid Multiple herbicide resistance. Xanthium sp. (552 Trp to Leu) (Bernasconi et al., 1995) Single amino acid Multiple herbicide resistance. Hordeum vulgare (Lee et al., 2011) (653 Ser to Asp) Single amino acid Resistant to sulfonylurea. Lactuca sativa (197 Pro to His) Specific activity of ALS reduced less (Eberlein et al., 1999) sensitive to feedback inhibition Single amino acid Chlorsulfuron resistance. Arabidopsis thaliana (197 Pro to Ser) Csr1-1 allele from mutant locus csr1 (Haughn et al., 1988) Single amino acid Sulfonylurea resistance. Nicotiana tabacum (196 Pro to Gln) C3 mutation of SuRA gene. (Lee et al., 1988) Double amino acid Sulfonylurea resistance. Nicotiana tabacum substitution S4-Hra mutation of SuRB gene. (Lee et al., 1988) (196 Pro to Gln and 573 Trp to Leu) Single amino acid Highly resistant (Tested on Nicotiana tabacum (573 Trp to Leu) chlorsulfuron only) (Kochevenko and Willmitzer, 2003) Single amino acid Highly resistant (Tested on Nicotiana tabacum ( 196 Pro to Ala, chlorsulfuron only) (Kochevenko and Willmitzer, 2003) Thre, Glut or Ser) Single amino acid Resistant (Tested on chlorsulfuron only) Nicotiana tabacum ( 196 Pro to Leu) (Harms et al., 1992; Kochevenko and Willmitzer, 2003) Single amino acid Multiple herbicide resistance. Nicotiana tabacum ( 255 Lys to Phe) (Yoon et al., 2002) Single amino acid Multiple herbicide resistance. Nicotiana tabacum ( 350 Met to Cys) Enzyme more sensitive to pH change (Le et al., 2003) Single amino acid Multiple herbicide resistance. Nicotiana tabacum ( 569 Met to Cys) (Le et al., 2003) Single amino acid Multiple herbicide resistance. Nicotiana tabacum ( 121 Ala to Thr) (Chong and Choi, 2000) Single amino acid Valine resistance. (Hervieu and Vaucheret, 1996) ( 214 Ser to Leu)

Isolation of mutants has been achieved through selection with and without mutagenesis and through site directed mutagenesis. Typically, resistance to ALS-inhibiting herbicides is due to gene mutation rather than amplification, duplication or changes in regulation. By selection in cell culture, Harms et al., (1992) were able to select a tobacco line resistant to sulfonylurea herbicides in which ALS was amplified 20 fold. However, they also demonstrated that this gene was mutated. Frequency of mutation to the ALS inhibiting enzymes is much higher than for glyphosate (Jander et al., 2003). Jander et al. (2003) estimated that in arabidopsis only 50,000 mutations are required to have a 95% probability of generating an ALS-inhibitor resistant line. Characterization of mutations In Lolium there is some evidence for resistance mechanism involving increased herbicide metabolism (Christopher et al., 1992) but in all other cases resistance to ALS-inhibitors is due to Copies of literature review single, and sometimes double, amino acid substitutions (Table 1). These mutations are thought to alter the herbicide binding site, and the enzymes consequently retain normal function. Multiple herbicide resistance as a result of a single mutation suggests an overlapping domain essential for herbicide binding (Hattori et al., 1995). In different species, amino acid substitutions or mutations at sites 193, 197 and 205 confer resistance to ALS inhibitors (White et al., 2003) The review by Tan et al., (2005) provides an overview of ALS-inhibiting mutations conferring resistance to imidazolinone herbicides in plants and more detail is also available in Duggleby and Pang (2000) and Duggleby et al. (2008).

Feedback inhibition in ALS mutants In the majority of cases, the enzymes of ALS-inhibiting mutants display normal feedback kinetics. However, in lettuce Eberlein et al., (1999) showed that the levels of valine, leucine and isoleucine were higher in seeds and leaves from herbicide resistant lines, suggesting altered feedback sensitivity. Plants of the csr1-4 double mutant line of Arabidopsis also displayed altered feedback inhibition and were less sensitive to added valine and leucine (Mourad et al., 1995).

ALS gene mutations in weed species As a result of the ease of mutation for resistance, and the selection pressure exerted by widespread use of ALS-inhibiting herbicides, resistant weed species have now been identified in the wild (Yu et al., 2010). Examples are given in Table 2.

Table 2. Examples of resistance mutations to ALS-inhibiting herbicides in weeds Species Amino acid Reference Type of resistance substitution Xanthium Trp552leu Bernasconi et al., 1995 Multiple Tranel et al., 2004 Kochia scoparia Pro173Thr Guttieri et al., 1992 Chlorsulfuron Raphanus Tyr122Ala Han et al., 2012 Sulfonylurea raphanistrum (wild radish) Helianthus spp. Ala205Asp White et al., 2003 Multiple (common sunflower) Apera spica-venti Pro197Thr Massa et al., 2011 Multiple Bromus tectorum Pro197Ser Park and Mallory-Smith, 2004 Multiple Amaranthus Pro248Leu Sibony et al., 2001 Sulfonylurea retroflexus Amaranthus Leu574Try Maertens et al., 2004 Sulfonylurea and hybridus imidazolinone

As the evolution of resistance in weeds has occurred several times, in different species, it is likely the mutations do not impact significantly on either ALS activity or fitness cost (Yu et al., 2010).

1.6 S4-Hra; ALS gene mutation in tobacco

The mutation of the SuRB gene that has been used as the selectable marker gene in the transformation vector pCGP1991 is S4-Hra. This mutation was derived from a line (S4) isolated from a mutagenized haploid cell culture (Chaleef and Ray, 1984) selected on the sulfonylurea herbicide chlorsulfuron. A diploid herbicide resistant plant homozygous for a single dominant gene was regenerated, which was then subjected to further selection (leading to the double mutant line S4-Hra) for increased resistance to chlorsulfuron (Creason and Chaleff, 1988). Lee et al., (1988) characterized and cloned the S4-Hra mutant of the SuRB gene, showing there were three nucleotide Copies of literature review substitutions in the S4-Hra line, when compared to the wild type. One was silent and two lead to amino acid substitutions (196 Pro to Gln and 573 Trp to Leu). Full details of the characterization of the mutant S4-Hra are provided in Bedbrook et al., (1991). Tobacco lines resistant to ALS- inhibitors have been isolated by independent researchers, who showed amino acid substitutions at the same amino acid positions (Table 1).

1.7 Crops resistant to ALS-inhibiting herbicides

ALS mutations can also be selected in the important crop plants (Kleter et al., 2011). For example in corn, plants regenerated from cell cultures selected on imidazolinine herbicides were shown to be a series of mutations which conferred resistance to all or some of ALS-inhibiting herbicides tested (Newhouse et al., 1991).The relative ease with which plants resistant to ALS-inhibiting herbicides can be selected has therefore been exploited by plant breeders to develop herbicide resistant crop plants (Tan et al., 2005). There are now many other crops available and Table 3 provides a list of varieties registered in Canada, as an example. The website from which the information in Table 3 was taken (http://www.inspection.gc.ca/plants/plants-with-novel-traits/approved) provides extensive information on the safety of the crops in question, which includes transgenic varieties. The mutations in the commercial varieties listed in Table 3 are single or double amino acid substitutions.

Table 3. Examples of crop varieties resistant to ALS-inhibiting herbicides. Data extracted from the Canadian Food Inspection Agency website Product Proponent Decision Date Imidazolinone resistant maize (3417R) Pioneer Hi-Bred May 30, 1994 International Imidazolinone resistant canola (lines NS738, Pioneer Hi-Bred April 25, NS1471, NS1473) International 1995 Sulfonylurea tolerant flax - CDC Triffid Crop Development February 16, Centre, University of 1998 Saskatchewan Imidazolinone tolerant rice lines CL121, CL141, and BASF Canada Inc. February 11, CFX51 (CLEARFIELD™ rice) 2002 Imidazolinone Tolerant CLEARFIELD™ Wheat BASF Canada March 21, (AP602CL) 2003 CLEARFIELDTM Imidazolinone tolerant lentil BASF Canada June 25, 2004 (RH44) CLEARFIELDTM Sunflower line X81359 BASF Canada October 25, 2005 DD2006-60: Determination of the Safety of BASF's BASF Canada June 22, 2006 Imidazolinone-Tolerant CLEARFIELD™ Wheat Events BW255-2 and BW238-3 DD2007-64: Determination of the Safety of BASF's BASF Canada January 4, Imidazolinone-Tolerant CLEARFIELD™ Durum 2007 Wheat Events DW2, DW6, and DW12 DD2008-69: Determination of the Safety of Pioneer Pioneer Hi-bred January 2, Hi-Bred Production Ltd.'s Sulfonylurea - Tolerant 2008 ExpressSun™ Sunflower (Helianthus annuus L.) SU7 DD2009-77: Determination of the Safety of Pioneer Pioneer Hi-bred August 26, Hi-Bred Production Ltd.'s Soybean (Glycine max 2009 (L.) Merr.) Event 356043 Copies of literature review

DD2010-80: Determination of the Safety of BASF BASF Canada June 4 2010 Canada Inc.'s Sunflower Line CLHA-PLUS and CL Sunflower Hybrid H4

2. An assessment of the potential toxicity of the ALS protein from the ALS gene S4-Hra used in the transformation vector pCGP 1991

2.1 Plant toxins

The chemical nature of plant derived compounds toxic to animals and man is extremely diverse, and potential toxicity is determined by dose, exposure time, the age and general health of the animal or person exposed, the combination of toxins and the way the plant is prepared before being consumed. The majority of plant toxins are non-protein secondary products, such as alkaloids and glycosides of alkaloids (Goetz et al., 2006). Plants may also accumulate minerals such as selenium and calcium oxalate to very high concentrations, making the plants toxic.

Some plant toxins are protein based. Excluding mushroom derived polypeptide toxins, the most toxic peptide based plant toxins are ribosome-inactivating proteins (Nielsen and Boston, 2001). This includes the lectins ricin, abris and concanavalin (Rudiger and Gabius, 2001) which are some of the most toxic compounds known to man. Lectins are largely found in beans and some grains. Two other groups of protein based toxins are; 1) Prolamins. These are storage proteins found in cereal seed and they cause toxicity symptoms in people suffering coeliac disease (McLachlan et al., 2002). 2) Thionins. These are low-molecular weight polypeptides that are found in the Gramineae (Bohlmann and Apel, 1991) and some other species (Romagnoli et al., 2000; Fung et al., 2003).

There are many databases and textbooks describing toxic plants The one we refer to here is from Indiana, USA (Goetz et al., 2006), which provides a rank of toxicity and identifies the chemicals responsible for toxicity, where this is known. This reference source identifies the following plants poisonous to animals, where the causative agent is protein based; • Abrus precatorius. The toxins in this plant are the protein abris, and one seed has sufficient protein to kill an adult human. • The Aroid family (i.e. Anthurium and Dieffenbachia). These plants contain proteolytic enzymes which release histamine and kinins. • Ricinus communis. The toxin in castor bean is ricin, a water soluble protein • Pteridium aquilinum The toxin in bracken fern is thiaminase, an enzyme that destroys thiamine (Kenton, 1957). • Equisetum arvense and Equisetum hyemale. The cause of toxicity is also thiaminase.

Toxicity of the ALS protein None of the peptide based classes of plant toxins described above are functionally or structurally related to the ALS gene product, and the ALS gene product can not be implicated as a cause of toxicity. Furthermore, classes of known plant peptide toxins are localized in seed or associated with cell walls, while ALS is not.

Mathesius et al. (2009) showed in a BLASTP search that the protein from the gm-hra gene (a modified soybean ALS gene with mutations analogous to those of SuRB) did not have sequence similarity to know toxic proteins. Mathesius et al. (2009) also isolated and purified the protein from the gm-hra gene and measured the toxicity of the protein in an acute toxicity assay in mice. The study showed the gm-hra protein was not toxic. Copies of literature review

2.2 The toxicity of tobacco

The toxicity of smoked and chewed tobacco to man is very well known. In fresh tobacco plants consumed by animals the toxin is nicotine. Nicotine is an alkaloid and is produced on a biosynthetic pathway unrelated to branched chain amino acid biosynthesis.

Tobacco can also cause occupational dermatitis (McBride et al., 1998; Mitchell and Rook, 2006). Tobacco workers are exposed to tobacco during processing, when leaves are harvested and dried. Contact is through touch and through tobacco dust carried in the air (Mitchell and Rook, 2006). Many other chemicals are used in tobacco processing and these may also contribute to, or be responsible for some dermatological reactions. Dermatitis in the tobacco industry is uncommon (Mitchell and Rook, 2006). However, nicotine is water soluble, and long term exposure to wet tobacco leaves can cause nicotine poisoning (so-called “green tobacco disease”, see McBride et al., 1998).

The ALS gene product can not be implicated as a cause of toxicity in tobacco.

2.3 Exposure to carnation

An important consideration in assessing the potential toxicity of the S4-Hra protein is a consideration of possible routes of contact. For carnation flowers imported into Europe the only realistic route of exposure is through handling.

Unlike an allergic response, which requires sensitization, toxin-mediated contact urticaria (chaffing, blistering, rash etc.) is not selective and occurs to most people with whom a plant may come into contact. McGovern and Barkley (2006) provide a detailed summary of plants causing urticara as a result of toxins. Most plants belong to the Urticaceae (nettle) family, but also the Hydrophyllaceae, Loasaceae and Euphorbiaceae. Common toxins include histamine, acetylcholine, and 5- hydroxytryptamine. Phytophotodermatitis disease (excessive darkening of the skin) is caused by the presence of furocoumarins such as bergapten and xanthotoxin (McGovern and Barkley, 2006). Tobacco is not a known source of any of these toxins, and neither ALS, nor the products of ALS, is involved in their biosynthesis.

Dermatological response to plants can also be through mechanical (thorns or oxalate crystals for example) and chemical irritants. There is no evidence that tobacco or ALS are chemical irritants (McGovern and Barkley, 2006).

2.4 Conclusions

There is sufficient evidence to suggest that the ALS gene product S4-Hra is not toxic. 1. ALS is a well conserved protein found in all plants, and S4-Hra is functionally identical to the numerous ALS enzymes found in plants and bacteria in nature. 2. Single amino acid substitution in ALS mutations conferring herbicide resistance are found in several food crops and weed species and occur naturally in bacteria. Humans eating plant food would have been exposed to a wide variety of enzymes very similar (even the same amino acid change) to the protein encoded by S4-Hra. Copies of literature review

3. ALS activity in widely grown imidazolinone-tolerant food crops is not biologically significant to parental varieties; the ALS enzyme still has the same physicochemical properties and functional activity. 4. ALS mutations, including S4-Hra, have been assessed as non-toxic by US and Canadian regulatory agencies. 5. There is no evidence that amino acid metabolism is affected in ALS mutant lines. 6. None of the known peptide based classes of plant toxins are functionally or structurally related to the ALS protein. 7. Tobacco is not a known source of phytophotodermatitis causing toxins, and neither ALS, nor the products of ALS, is involved in the biosynthesis of compounds which are.

The S4-Hra gene product in carnation will not, in any event, be consumed as a food. The transgenic carnation has a long history of large-scale, safe use, with no reports of any toxic effects.

3. An assessment of the potential allergenicity of the ALS protein from the ALS gene S4-Hra used in the transformation vector pCGP1991

3.1 Plant allergens

Allergens of plant origin are found in food and drinks, in the atmosphere (pollen and dust borne plant particles) and in situations where direct contact is made with plant or processed plant material. Allergens are usually proteins (Mills et al., 2004), but not always (Table 5)

Table 5. Plant families with the most commonly encountered contact allergens (extracted from McGovern and Barkley, 2006) Family Examples Allergen Anacardiaceae Poison Ivy. Poison oak, Catechols and resorcinols (1,2 and 1,3-- dihydroxybenzenes) Anacardiaceae Mango skin, cashew husk Phenols and resorcinol Ginkgoaceae Gingko seed pulp Anacardic acid, Proteaceae Grevillea Pentadecylresorcinol Compositae Chrysanthemum, dandelion Sesquiterpene lactones (Asteraceae) ragweeds, worts etc. Compositae Chicory, sunflower Lactucin and lactopicrin, 1-0-methyl 1-4,5- (Asteraceae) dihydroniveusin A Jubulaceae Liverwort, magnolia Sesquiterpene lactones Alliaceae Garlic, onion Diallyl disulfide, allylpropyl disulfide, and allicin Alstroemeriaceae Alstromeria Tuliposide A Liliaceae Tulips Tuliposide A Primulaceae Primula Primin

Most food allergens are a result of cross-reaction; the result of initial contact with allergens that sensitizes individuals to similar allergens later found in food. Allergens may be initially contacted through inhalation, of pollen for example (van Ree, 2002), or touch. The Hev series of allergens in natural rubber latex, for example, cause cross reactivity to a large number of fruits and vegetables (Wagner and Breitender, 2002). True food allergens capable of sensitization after ingestion typically tolerate low pH and proteolytic degradation (Mills et al., 2004).

There a large number of characterized allergens, which have been grouped into families based on the structural and functional similarities of the proteins. Groups of plant food allergens have been summarized by Mills et al. (2004) and Jenkins et al. (2005). Major groups include the prolamins, Copies of literature review profilins, Bet v 1 family, Lol p1 family, papain family and cupins. The profilins are also a major group of food allergens that also occur in pollen and natural rubber latex, along with pathogenesis related proteins such as chitinases and glucanases, which may also be allergens (Wagner and Breitender, 2002).

A large number of pollen allergens have now been cloned (de Weerd et al., 2002). As a major source of health problems grass pollen allergens are a well studied group and have been grouped by function (Andersson and Lidholm, 2003; see summary Table 6).

Table 6. Immunological classification of major grass pollen allergen groups (Andersson and Lidholm, 2003) Group Comment Allergen characteristic 1 Major allergen. 90% of sensitized Glycoproteins individuals are cross reactive 2,3 Minor allergen. Lolium and Dactylis Non-glycosylated proteins of 95 -98 amino acids 4 Major allergen. 80% of sensitized Highly basic glycoproteins. Possibly individuals are cross reactive flavoproteins. 5 Dominant allergen in Poodieae family. Glycoproteins, similar to group 1 6 Poa species only. 60 - 70% of sensitized Acidic protein of ca. 13kD individuals are cross reactive 7 Also found in Brassica, olive and birch Small proteins of ca. 8o amino acids 11 Lolium Similar structure to soybean trypsin inhibitor 12 Minor allergens in grasses Profilins 13 Phleum pratense Probable polygalacturonase

Radauer and Breiteneder (2006) have recently provided a summary of pollen allergens, grouped by abundance and taxonomy. The families with the most allergens were the grasses (Poaceae), followed by the Cupressaceae. The tobacco family (Solanaceae) was not represented in the survey. The most important classes of allergens were identified as the prolamins (Shewry et al., 2002). These are a large group of sulphur-rich seed proteins, including trypsin/ α-amylase inhibitors and lipid transfer proteins (van Ree et al., 2002). Other classes of allergens listed were the expansins, which are common in grass allergens (Andersson and Lidholm, 2003), profilins (also found in natural rubber latex) and thaumatin-like proteins. Thaumatin-like proteins are a class of pathogenesis-related proteins (Breitender, 2004). Other examples of protein allergens found in pollen are EF-hand, Ole e1-like, pectate lyases and ribonucleases.

The ALS protein ALS has never been identified as an allergen despite the presence of this protein in all plants. A search of the plant food-allergens, aero-allergens and contact allergens in the Faarp protein database of known allergens (www.allergenonline.com) did not list ALS from any source. Mathesius et al. (2009) isolated and purified the protein from the gm-hra gene (a modified soybean ALS gene with mutations analogous to those of SuRB) and showed that the protein was quickly degraded by the digestive enzymes pepsin or pancreatin. The same authors demonstrated that enzymatic activity was lost at temperatures above 50ºC. These characteristics are not usually associated with a protein food allergen (Mills et al., 2004).

3.2 Allergenicity of tobacco

Cases of hypersensitivity to tobacco are cited in Mitchell and Rook (2006). Tobacco pollen has been shown to contain an allergenic profilin (Mitterman et al., 1995). A search of the aero-allergens Copies of literature review in the Faarp protein database of known allergens (www.allergenonline.com) identified Calcium- binding proteins, villin 1 and 2 and Beta expansin-like protein from Nicotiana tabacum. None of these are functionally related to ALS.

The antigen in tobacco leaf is cross-reactive to mugwort pollen (Ortega et al., 1999; Armentia et al., 2005), tomato extract (Ortega et al., 1999) and Lolium pollen (Armentia et al., 2005). There is no evidence that the ALS gene product is an antigen in tobacco.

3.3 Allergens in flowers

Carnation is not a food. Carnation flowers imported into Europe the only realistic route of exposure to potential allergens is therefore through handling.

Along with food processors, florists and flower growers have one of the highest rates of occupational asthma as they experience long-term, high-duration exposure to allergens in pollen through handling plant material and through dust (de Jong et al., 1998; Goldberg et al., 1998; Akpinar-Elci et al., 2004; Emberlin et al., 2004; Mapp et al., 2005). In a comparison of different types of European crop growers Monso et al. (2000) identified flower growing as a high risk factor for asthma. In situations where large numbers of flowers are grown or sold there are also secondary sources of allergens. This can include insect allergens from pest and predatory mites (Cistero- Bahima et al., 2000; Johnasson et al., 2003) and fungal allergens such as Aspergillus (Monso et al., 2002; Emberlin et al., 2004). Sensitizing chemicals are also found in the florist’s workplace. Allergenicity to flowers is a minor medical problem. Symptoms usually disappear away from the workplace De Jong (1998) and change of profession offers a permanent solution (Goldberg et al., 1998; Monso et al., 2002). The high intensity exposure by florists may also result in sensitization not seen in the general population (Bolhaar and van Grinkel et al., 2000).

Flowers of the Compositae (Asteraceae), such as chrysanthemum (De Jong et al., 1998; Groenewoud et al., 2002) are known to be major sources of aero-allergens. Other species are listed in Table 7. Carnation is insect pollinated and any pollen produced is not wind-borne.

Table 7. Flower species known to exhibit air-borne allergenicity Species Reference Species Reference Chrysanthemum Monso et al., 2002 Narcissus Monso et al., 2002 leucanthemum pseudonarcissu Solidago Monso et al., 2002 Hyacinthus Monso et al., 2002 canadiensi orientalis Helianthus Monso et al., 2002 Molucella Miesen et al., 2003 annuus Gladiolus Monso et al., 2002 Easter lily Piiriala et al.,1999 spp. Stephanotis Van der Zee et al., Rosa rugosa Demir et al., 2002 floribunda 1999

Emberlin et al. (2004) identified cyclamen, solidago, lisianthus, lily, hypercium and alstromeria pollen as the most abundant in flower shops.

The causative agents of the allergic reactions to flowers are largely unidentified, but where known does not include the ALS gene product (Table 8).

Copies of literature review

Copies of literature review

Table 8. Adverse contact reactions through handling floricultural products. Plant Allergen Reference Bishops weed Psoralens Kiistala et al., 1999 Easter lily Not identified Piiriala et al.,1999 Tulip Tuliposide-A Piiriala et al.,1999 Hydrangea Hydrangenol Rademaker, 2003 Alstromeria Tuliposide-A Kristiansen and Christense, 1998 Carnation IgE bands at 15 and 17kDa Vidal and Polo, 1998 Lily IgE bands at 19 and 22 kDa Vidal and Polo, 1998 Chrysanthemum arteglasin-A McGovern and Barkley, 2006 Lilium profilins Faarp protein database; DeWeerde et al., 2002

3.4 The presence of contiguous amino acid sequences in S4-Hra homologous to sequences in known allergens

Kleter and Pejnenburg (2002) showed that a 6 contiguous amino acid sequence in the S4-Hra gene shares homology with a single known allergen, the Amb a 1.4 from the pollen of ragweed, Ambrosia artemisiifolia. Amb a 1.4 is a minor allergen in ragweed, most likely encoding a pectate lyase (Wopfner et al., 2005).The short amino acid sequence (PRKGSD) identified by Kleter and Pejnenburg (2002) occurs at positions 96 to 101 in tobacco (Lee et al., 1988) and so does not include the two amino acid substitutions that distinguish S4-Hra from the wild type ALS gene SuRB. Both SuRA and SuRB therefore also carry this motif (Lee et al., 1988). The 6 amino acid sequence also excludes the herbicide binding site. Because ALS is a relatively well conserved gene, a very large number of other plants therefore most probably have the same contiguous sequence of 6 amino acids. A BLAST search identified it in sunflower, maize and rice. In several species where there is not an exact match to the sequence PRKGSD, it is consistently matched to PRKGAD. This includes certain maize varieties (Fang et al., 1992), cotton (Grula et al., 1995), sunflower and a herbicide resistant line of Sinapis arvensis. Mathesius et al. (2009) showed the protein from the gm- hra gene (a modified soybean ALS gene with mutations analogous to those of SuRB) did not have sequence similarity to know allergenic proteins.

Recognizing the limitations of sequence comparison alone, Kleter and Pejnenburg (2002) employed an additional screen on positive matches, the hydrophilicity plot of Hopps and Woods. Proteins (either the target protein or the homologous allergen) where the region of maximum hydrophilicity coincided with the 6 amino acid sequence would then qualify for further examination as potential allergens. In the case of ALS, an allergen-homologous sequence (KVLENR) that was found to occur in the ALS from arabidopsis (Kleter and Pejnenburg, 2002) fell into this category. The sequence (KVLENR) does not occur in S4-Hra (Lee et al., 1988 and BLAST search, Attachment A8). It does however, occur in the ALS of the food crop Brassica napus (Bekkaoui et al., 1991).

The sequence PRKGSD was not sufficiently hydrophilic to be indicative of an allergen (Kleter and Pejnenburg (2002). As discussed above, the related sequence, PRKGAD (a serine to alanine substitution from PRKGSD), also occurs in many food crops. This substitution would reduce the total hydrophilicity of the 6 amino acid sequence from +9.3 to +8.8 (Hopp and Woods, 1981), meaning it also is not sufficiently hydrophilic.

Amino acid sequence as a predictor of allergenicity Sequence comparison is one tool for screening for potential allergens in proteins. In addition to the work of Kleter and Pejnenburg (2002), comparison programs have been published by Riaz et al. (2005), Stadler and Stadler (2003) and Bjorkland (2005). These authors have criticized the use of 6 contiguous amino acids as an indication of potential allergenicity, as recommended by the Copies of literature review

FAO/WHO (FAO/WHO, 2001). Stadler and Stadler (2003) found a very high percentage of “false positives” using such a technique, with two-thirds of all proteins in the Swiss-prot database determined to be allergens, and recommended the use of allergen motifs for screening. Hileman et al. (2001) concluded the use of 6 amino acid windows produced many irrelevant matches, and this was also acknowledged by Kleter and Pejnenburg (2002). The amino acid sequence may have no prediction value without knowledge of secondary protein structure, such as the case with B-type epitopes (Bjorkland et al., 2005).

Lack of sequence similarity does not mean a protein can never be an allergen and short commonality of sequence is not necessarily related to similar function. In fact, there is ample evidence that sequence similarity does not necessarily translate to structural similarity of a folded protein because sequence differences do not generate predictable structural differences. Sequence alone does not provide any information on possible post translational modifications. The link between antigenicity and allergenicity and the presence of certain motifs is tenuous, because it would suggest that more allergens would be found and could readily be engineered. In the case of the double amino acid substitution in S4-Hra, if the protein then became an allergen there would be many similar allergens in many, many plants, and it is very likely that they would have been identified by now if they existed. It might also be expected that if S4-Hra was an allergenic protein far more homologies with sequences in other allergens (the entire Amb series for example), might have been identified.

There are many proteins that have strong characteristics of an allergen, including sequence homology, but do not elicit an allergenic response (Stadler and Stadler, 2003; Lehrer and Bannon, 2005).

Factors in determining whether a protein is allergenic or not, that should be considered in addition to sequence homology include; • A high number of disulphide bridges, conferring stability (Mills et al., 2004) • For true food allergens, high concentration, enhancing the probability of protein resistance to digestion • Ability to bind to membranes (Mills et al., 2004) • Proteolytic activity of the allergen (Jenkins et al., 2005) • Three dimensional structure, including folding, tunnels and total surface area, of the mature protein (Aalberse et al., 2000; Jenkins et al., 2005). The conservation of specific surface residues and main chain conformations was found by Jenkins et al. (2005) to play an important role in conservation of IgE-binding epitopes.

3.5 Conclusions - Allergenicity of the S4-Hra mutant SuRB gene

There are strong reasons to suggest that the S4-Hra gene does not encode an allergen. 1. The ALS protein is highly conserved and S4-Hra is very similar to the protein found in all plants. Though the sequence, and so degree of variation, of native carnation ALS is unknown it is likely the sequence of S4-Hra is not significantly different to the sequences endogenous to carnation or to those found in a very wide range of plant foods and other plants. 2. The ALS protein is ubiquitous, but there is no evidence of cross-reactivity as a result of sensitization to the ALS protein 3. Short amino acid sequences from ALS, with homology to sequences in an allergen, are known to occur in some plants, and probably occur in many others, yet there is no evidence the ALS protein is allergenic. a. Wild type tobacco has exactly the same short amino acid sequence but allergenic response in tobacco is not due to ALS. Copies of literature review

b. The short amino acid sequence has insufficient hydrophilicity to warrant further study as an allergen (Kleter and Pejnenburg, 2002). 4. ALS mutations similar to S4-Hra are now present in many food crops and the pollen of weed species. However, ALS has never been identified as an allergen despite this widespread opportunity for exposure. a. ALS mutations in food crops, including S4-Hra, have been assessed as non- allergenic by US, Australian and Canadian regulatory agencies. 5. Allergens are typically abundant and stable (Mills et al, 2004). ALS is not found at high concentration and is not stable. The ALS protein is heat labile and sensitive to trypsin and highly unlikely to be a true food allergen 6. ALS proteins are not structurally or functionally similar to any known allergen groups in food or pollen. 7. An important consideration in assessing the potential allergenicity of the S4-Hra gene product is also a consideration of possible routes of contact. Carnation is not a food and does not produce wind borne pollen, so in carnation flowers imported into Europe the only realistic route of exposure is through skin contact during handling within the floral industry. a. Occupationally, the people most likely to be regularly exposed to the carnation carrying the S4-Hra gene are florists. Allergies in this work group have never been attributed to the ALS protein or structurally or functionally similar proteins

The genetically modified carnations are not a food, and have not been assessed for food or animal use by any other regulatory body that has approved the commercial release of the flowers. It is also unrealistic to expect that any of the imported carnation flowers will be adopted for use as food or animal feed. The S4-Hra gene product in carnation will therefore not, in any event, be consumed as a food.

4. Biosafety evaluation of food crops containing ALS gene mutations

4.1 Canadian assessments of imidazolinone-tolerant Clearfield™ crops

As representative examples, we have reviewed the decision documents for three imidazolinone-tolerant Clearfield™ crops; rice, wheat and sunflower (Canadian Food Inspection Agency, 2002, 2004, 2005), all of which were single amino acid substitution mutations for ALS. Though not transgenic, the Canadian government assesses the varieties for food safety in a similar way to transgenic varieties. The following conclusions were specific to the ALS gene; • Regulation of the levels of valine, leucine and isoleucine by feedback inhibition was not affected in any of the three varieties. • Unlike known food allergens, ALS is a minor protein in plant tissue (~0.001% of total protein in rice seed) and is heat sensitive and trypsin susceptible. Specific data is given in Table 9.

Table 9. Stability of ALS from imidazolinone-tolerant varieties of three species Time for complete loss of activity Sunflower Wheat Rice Heat sensitivity 1 min at 100ºdegree 1 min at 100ºdegree 1 min at 100ºdegree Trypsin 60 min 30 min 5 min

• The unmodified form of the ALS protein in all three species showed no amino acid similarity to known allergens. • ALS from none of the three species is a known toxin or allergen. Copies of literature review

• Difference in levels of ALS activity in imidazolinone-tolerant and imidazolinone- susceptible leaf tissue is not biologically significant as the ALS enzyme in both still has the same physicochemical properties and functional activity.

4.2 Food crops containing the SuRB gene mutation S4-Hra

To our knowledge, no food crop variety has been commercialized in which the SuRB gene mutation we have used in the carnation line FLO-40685-1 has been used in the transformation vector. However, the gm-hra gene, a modified soybean ALS gene, has mutations analogous to those of SuRB, and transgenic plants containing gm-hra have been studied in several animal feeding studies (see section 4.4). The results of other studies concluded the protein derived from gm-hra poses no safety issues (Mathesius et al., 2009).

A transgenic cotton line resistant to sulfonylurea was developed, but never commercialized. This line was developed by DuPont, who did prepare a petition for deregulation to the US government (DuPont, 1995). In that petition, levels of cotton toxicants were measured, but no direct measurements of the toxicity or allergenicity of the ALS gene or ALS enzyme were included (that data is included in a petition to FDA, which we do not have). It was pointed out in the petition that; • Even though herbicide resistant, the ALS gene was still functionally identical to the numerous ALS genes found in plants and bacteria in nature. • ALS enzymes tolerant to sulfonylurea herbicides occur in bacteria naturally. In reviewing the application USFDA (1996) took into consideration, given the high homology in ALS, that animal and humans eating plant food would have been exposed to a wide variety of very similar enzymes. The University of Queensland has developed a transgenic pineapple line using the S4-Hra gene as the selectable marker. The potential toxicity and allergenicity of the gene was reviewed by the Australian government (OGTR, 2003) who concluded there was no potential risk on the basis that the enzyme is not a known toxin or allergen and related enzymes are expressed in a variety of edible plants (e.g. soybean and rice). The review also pointed out the high homology to ALS from other species, and lack of sequence homology with known allergens.

Arabidopsis carrying the csr1-2 gene had a significant increase in transcript level but this was the most significant difference in the transcriptome revealed by microarray analysis. There were no other significant pleiotropic effects on gene expression (Schnell et al., 2012).

4.3 Transgenic flax

The csr1-1 gene from arabidopsis, which confers resistance to sulfonylurea herbicides, has been used as a selectable marker in a number of transgenic plants, and in the development of a flax variety resistant to these herbicides. Agronomically, the transgenic flax line is not significantly different to the parent (McSheffrey et al., 1992). In the USFDA response to a petition for use as animal feed and food (USFDA, 1998) it was stated that the ALS activity was 56.3 nanomoles (nmol)/mg/hr for the parent cultivar compared to 88.8 nmol/mg/hr in the transgenic. The difference was attributed to the combined activity of the endogenous and inserted ALS, not altered feedback inhibition. There was no difference in seed amino acid composition between the two lines.

4.4 Animal feeding studies

Table 10 summarizes the conclusions from animal feeding studies in which seed and extracts from transgenic plants containing modified ALS genes have been compared to appropriate non- transgenic control lines. All these studies concluded there was no difference between the two sources of animal feed on animal weight or animal health. Copies of literature review

Table 10. Animal feeding studies in which seed and extracts from transgenic plants containing modified ALS genes have been compared to appropriate non-transgenic control lines. Reference Test Source Characterization Conclusion ALS gene or animal protein Appenzeller Rat Soybean Body weight, clinical No biologically gm-hra et al., 2008 signs, mortality, sensory relevant adverse effects response, clinical pathology, organ weights, gross pathology, microscopic pathology Appenzeller Rat Maize Body weight, No biologically zm-hra et al., 2009 Ophthalmology, relevant adverse effects Neurobehavioral evaluation, clinical pathology, gross and anatomical pathology Chukwudbe Rat Soybean Body weight, organ No substantial csr 1-2 et al., 2012 weights, Clinical alteration to pathology, gross necropsy composition of and histopathology soybeans. No adverse effects on nutritional or safety status. Delaney et Rat Soybean Body weight, clinical No biologically gm-hra al., 2008 signs, mortality, toxicity, relevant adverse effects sensory response, clinical pathology, organ weights, gross pathology, clinical pathology Mathesius Mice Acute Body weight and lesions at No significant Purified et al., 2009 toxicity necropsy differences. protein from test gm-hra gene administered at 463 mg/kg body weight Mathesius Mice Repeated Body weight, clinical and No biologically Purified et al., 2009 dose ophthalmological relevant adverse effects protein from toxicity observations, motor gm-hra gene assay activity, clinical chemistry administered response, histopathological in food at observations 1000 mg/kg body weight McNaughton Chicken Maize and Weight and egg No significant zm-hra, gm- et al., 2011a Soybean production, cracking differences. hra McNaughton Chicken Maize and Weight gain, feed intake, No significant zm-hra, gm- et al., 2011b Soybean carcass yield differences. hra Meija et al., Chicken Soybean Weight and egg production No significant gm-hra 2010 and quality, cracking differences.

5. Overall conclusions

Copies of literature review

A review of the literature indicates that the SurB gene product is non-toxic and non-allergenic. This conclusion is supported by experimental evidence from research using related ALS proteins (Mathesius et al., 2009). It may be considered that the SurRB ALS is substantially equivalent to the naturally occurring ALS enzymes found in all plants .The transgenic carnation now has a long history of large-scale, safe use with no reports of any toxic or allergenic effects.

6. Literature cited

1. Aalberse, R.C. Structural biology of allergens. Journal of Allergy and Clinical Immunology 106: 228–238, 2000. 2. Akpinar-Elci, M., Elci, O.C. and Odabast A. Work-related asthma-like symptoms among florists. Chest 125: 2336–2339, 2004. 3. Andersson, K. and Lidholm, J. Characterisation and immunobiology of grass pollen allergens. International Archives of Allergy and Immunology 130: 87–107, 2003. 4. Appenzeller, L.M., Munley, S.M., Hoban, D., Sykes, G.P., Malley, L.A. and Delaney, B. Subchronic feeding study of herbicide-tolerant soybean DP-356043-5 in Sprague-Dawley rats. Food and Chemical Toxicology 46: 2201 – 2213, 2008. 5. Appenzeller, L.M., Malley, L., Hoban, D., Sykes, G.P. and Malley, L.A.. Subchronic feeding study of grain from herbicide-tolerant maize DP-098140-6 in Sprague-Dawly rats. Food and Chemical Toxicology 47: 2269 - 2280, 2009. 6. Armentia, A., Baertolome, B., Puyo, M., Pardo, M., Calderon, S., Asensio, T., Martin, G., Fernandez, S., Hernandez, M. and Gonzalex-Sagrado, M. El tabaco como alergeno en enfremedad bronquial obstructive. Estudio preliminar. Alergol.Immunol.Clin. 20: 14–27, 2005. 7. Bedbrook, J.R., Chaleff, R.S., Falco, S.C., Mazur, B.J., Somerville, C. and Yadav, N.S. Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase. US patent number 5,013,659, 1991. 8. Bekkaoui, F., Condle, J.A., Neustaedter, D.A., Moloney, M.M. and Crosby, W.L. Isolation, structure and expression of a cDNA for acetolactate synthase from Brassica napus. Plant Molecular Biology 16: 741–744, 1991. 9. Bernasconi, P., Woodworth, A.R., Rosen, B.A., Subramanian, M.V. and Siehl, D.L.A naturally occurring point mutation confers broad range tolerance to herbicides that target acetolactate synthase. Journal of Biology Chemistry 270: 17381–17385 1995. 10. Bjorklund, A.K., Soeria-Atmadja, D., Zorzet, A., Hammerling, U. and Gustafsson, M.G. Supervised identification of allergen-representative peptides for in silico detection of potentially allergenic proteins. Bioinformatics 21: 39–50, 2005. 11. Bohlmann, H. and Apel, K. Thionins. Annual Review of Plant Physiology 42: 227–240, 1991. 12. Bolhaar, S.T.H.P. and van Ginkel, C.J.W. Occupational allergy to cyclamen. Allergy 55: 418–419, 2000. 13. Breiteneder, H. Thaumatin-like proteins – a new family of pollen and fruit allergens. Allergy 59: 479-481, 2004. 14. Canadian Food Inspection agency. Decision document DD2002-40. Determination of the safety of BASF Canada’s imazethapyr-tolerant (Clearfield™) rice, 2002. 15. Canadian Food Inspection agency. Decision document DD2004-48. Determination of the safety of BASF Canada’s imidazolinone-tolerant (Clearfield™) wheat teal 11A, 2004. 16. Canadian Food Inspection agency. Decision document DD2005-50. Determination of the safety of BASF Canada imidazolinone-tolerant Clearfield™ Sunflower (Helianthus annus L.) Hybrid X81359, 2005. 17. Chaleff, R.S. and Bascomb, N.F. Genetic and biochemical evidence for multiple forms of acetolactate synthase in Nicotiana tabacum. Molecular and general genetics 210: 33–38, 1987. Copies of literature review

18. Chaleff, R.S. and Mauvais, C.J. Acetolactate synthase is the site of action of two sulfonylurea herbicides in higher plants. Science 224: 1443–1445, 1984. 19. Chaleff, R.S. and Ray, T.B. Herbicide-resistant mutants from tobacco cell cultures. Science 223: 1448-1511, 1984. 20. Chen, H., Saksa, K., Zhao, F., Qiu, J. and Xiong, L. Genetic analysis of pathway regulation for enhancing branched-chain amino acid biosynthesis in plants. The Plant Journal 63: 573 – 583, 2010. 21. Chukwudbe, A., Privalle, L., Reed, A., Wandelt, C., Contri, D., Dammann, M., Groeters, S., Kaspers, U., Strauss, V. and van Ravenzwaay, B. Health and nutritional status of Wistar rats following subchronic exposure to CV127 soybeans. Food and Chemical Toxicology 50: 956 - 971, 2012. 22. Chong, C-K. and Choi, J-D. Amino acid residues conferring herbicide tolerance in tobacco acetolactate synthase. Biochemical and Biophysical Research Communications 279: 462– 467, 2000. 23. Christopher, J.T, Powles, S.B. and Holtum, J.A.M. Resistance to acetolactate synthase- inhibiting herbicides in annual ryegrass (Lolium rigidum) involves at least two mechanisms. Plant Physiology 100: 1909–1913, 1992. 24. Cistero-Bahima, A., Enrique, E., Alonso, R., san Miguel, M.M. and Bartolome, B. Simultaneous occupational allergy to a carnation and its parasite in a greenhouse worker. Journal of Allergy and Clinical Immunology 106: 780, 2000. 25. Creason, G.L. and Chaleff, R.S. A second mutation enhances resistance of a tobacco mutant to sulfonylurea herbicides. Theoretical and Applied Genetics 76: 177–182, 1988. 26. DeJong, N.W., Vermeulen, A.M., van Wijk, G. and de Groot, H. Occupational allergy caused by flowers. Allergy 53: 204–209, 1998. 27. Delaney, B., Appenzeller, L.M., Munley, S.M., Hoban, D., Sykes, G.P., Malley, L.A. and Sanders, C. Subchronic feeding study of high oleic acid soybeans (Event DP-305423-1) in Sprague-Dawley rats. Food and Chemical Toxicology 46: 3808 – 3817, 2008. 28. Demir, A.U., Karakaya, G. and Kalyoncu, A.F. Allergy symptoms and IgE immuno response to rose; an occupational and an environmental disease. Allergy 57: 936–939, 2002. 29. DeWeerd, N.A., Bhalla, P. and Singh, M.B. Aeroallergens and pollinosis; molecular and immunological characteristics of cloned pollen allergens. Aerobiologia 18: 87–106, 2002. 30. Duggleby, RG and Pang, SS. Acetohydroxyacid synthase. Journal of Biochemistry and Molecular Biology 33: 1–36, 2000. 31. Duggleby, R.G., McCourt, J..A and Guddat, L.W. Structure and mechanism of inhibition of plant acetohydroxyacid synthase. Plant Physiol. and Biochem. 46: 309 – 324, 2008. 32. DuPont. Petition to USDA/APHIS for determination of non-regulated status; sulfonylurea tolerant cotton line 19-51A, 1995. 33. Eberlein, C.V., Guttieri, M.J., Berger, P.H., Fellman, J.K., Mallory-Smith, C.A., Thill, D.C., Baerg, R. and Belknap, W.R. Physiological consequences of mutation for ALS-inhibitor resistance. Weed science 47: 383–392 1999. 34. Emberlin, J., Adams-Grooms, B., Treu, R. and Carswell, F. Airborne pollen and fungal spores in florists shops in Worcester and in Bristol, UK: a potential problem for occupational health. Aerobiologie 20: 153–160, 2004. 35. Fang, L.Y., Gross, P.R., Chen, C-H. and Lillis, M. Sequence of two acetohydroxyacid synthase genes from Zea mays. Plant Molecular Biology 18: 1185–1187, 1992. 36. FAO/WHO. Evaluation of allergenicity of genetically modified foods. Report of a joint FAO/WHO expert consultation on allergenicity of foods derived from Biotechnology 22 – 25 January, 2001. 37. Fung, T.L., Brugliera, F. and Anderson, M.A. Isolation and properties of floral defensins from ornamental tobacco and petunia. Plant Physiology 131: 1283–1293, 2003. Copies of literature review

38. Goetz, R.J, Jordan, T.N., McCain, J.W. and Su, N.Y. Indiana plants poisonous to livestock and pets. Available on-line at http://www.vet.purdue.edu/depts/addl/toxic/cover1.htm, 2006. 39. Goldberg, A., Confino-Cohen, R. and Waisel, Y. Allergic response to pollen of ornamental plants: high incidence in the general atopic population and especially among flower growers. Journal of Allergy and Clinical Immunology 102: 210–214, 1998. 40. Groenewoud, G.C.M., De Jong, N.W., Burdorf, A., De Groot, H. and van Wyk, R.G. Prevalence of occupational allergy to Chrysanthemum pollen in greenhouses in the Netherlands. Allergy 57: 835–840, 2002. 41. Grula, J.W., Hudsperth, R.L., Hobbs, S. and Anderseon, D.M. organization, inheritance and expression of acetohydroxyacid synthase genes in the cotton allotetraploid Gossypium hirsutum. Plant Molecular Biology 28: 837–846, 1995. 42. Guttieri, M.J., Eberlein, C.V., Mallory-Smith, C.A., Thill, D.C. and Hoffman, D.L. DNA sequence variation in domain A of the acetolactate synthase genes of herbicide-resistant and –susceptible weed biotypes. Weed Science 40: 670–676, 1992. 43. Han, H., Yu, Q., Purba, E., Li, M., Walsh, M., Friesen, S. and Powles, S.B. A novel amino acid substitution Ala-122-Tyr in ALS confers high-level and broad resistance across ALS- inhibiting herbicides. Pest Manag. Sci 68: 1164 – 1170, 2012. 44. Harms, C.T., Armour, S.L., DiMario, J.J., Middlesteadt, L.A., Murray, D., Negrotto, D.V., Thopson-Taylor, H., Weymann, K., Montoya, A.L., Shillito, R.D. and Jen, G.C. Herbicide resistance due to amplification of a mutant acetohydroxyaacid synthase gene. Molecular and General Genetics 233: 427–435, 1992. 45. Hattori, J., Brown, D., Mourad, G., Labbe, H., Ouellet, T., Sunohara, G., Rutledge, R., King, J. and Miki, B. An acetohydroxyacid synthase mutant reveals a single site involved in multiple herbicide resistance. Molecular and General Genetics 246: 419–425, 1995. 46. Haughn, G.W., Smith, J., Mazur, B. and Somerville, C. Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonyulurea herbicides. Molecular and General Genetics 211: 266–271, 1988. 47. Hervieu, F. and Vaucheret, H. A single amino acid change in acetolactate synthase confers resistance to valine in tobacco. Molecular and General Genetics 251: 220–224, 1996. 48. Hileman, R.E., Silvanovich, A., Goodman, R.E., Rice, B.A., Holleschak, G., Astwood, J.D. and Hefle, S.L. Bioinformatic methods for allergenicity assessment using a comprehensive allergen database. International Archives of Allergy and Immunology 128: 280–291, 2002. 49. Hopp, T.P. and Woods, K.R. Prediction of protein antigenic determinants from amino acid sequences. Proceedings of the National Academy of Science 78: 3824–3828, 1981. 50. Jander, G., Baerson, S.R., Hudak, J.A., Gonzalez, K.A., Gruys, K.J. and Last, R.L. Ethylmethanesulfonate saturation mutagenesis in Arabidopsis to determine frequency of herbicide resistance. Plant Physiology 131: 139–146, 2003. 51. Jenkins, J.A., Griffith-Jones, S., Shewry, P.R., Bretender, H. and Mills, E.N.C. Structural relatedness of plant food allergens with specific reference to cross—reactive allergens: an in silico analysis. Journal of Allergy and Clinical Immunology 115: 163–170, 2005. 52. Johansson, E., Kolmodin-Hedman, B., Kallstrom, E., Kasier, L. and hage-Hamsten, M. IgE- mediated sensitization to predatory mites in Swedish greenhouse workers. Allergy 58: 337– 341, 2003. 53. Keeler, S.J., Saanders, P., Smith, J.K. and Mazur, B.J. Regulation of tobacco acetolactate synthase gene expression. Plant Physiology 102: 1009–1018, 1993. 54. Kenton, .RH. The partial purification and properties of a thiaminase from bracken (Pteridium aquilinum (L.) Kuhn). Biochemical Journal 67: 25–33, 1957. 55. Kiistala, R., Makinen-Kilunen, S., Heikkinen, K., Rinne, J. and Haahtela, T. Occupational allergic rhinitis and contact urticaria caused by Bishop’s weed (Ammi majus). Allergy 54: 635–639, 1999. Copies of literature review

56. Kleter, G.A. and Pejnenburg, A.A.C.M. Screening of transgenic proteins expressed in transgenic food crops for the presence of short chain amino acid sequences identical to potential, IgE-binding linear epitopes of allergens. BMC Structural Biology 2: 8, 2002. 57. Kleter, G.A., Unsworth, J.B. and Harris, C.A. The impact of altered herbicide residues in transgenic herbicide-resistant crops on standard setting for herbicide residues. Pest Manag Sci 67: 1193 - 1210, 2011 58. Kochevenko, A. and Willmitzer, L. Chimeric RNA/DNA oligonucleotide-based site-specific modification of the tobacco acetolactate synthase gene. Plant Physiology 132: 174–184, 2003. 59. Kristiansen, K. and Christensen, L.P. Allergen contents in Alstromeria can be reduced by breeding. Euphytica 101: 367–375, 1998. 60. Le, D.T., Yoon, M-.Y, Kim, Y.T. and Choi, J-D. Roles of conserved methionine residues in tobacco acetolactate synthase. Biochemical and Biophysical Research Communications 306: 1075-1082, 2003. 61. Le, D.T., Yoon, M-Y., Kim, Y.T., Choi, J-D. Roles of three well-conserved arginine residues in mediating the catalytic activity of tobacco acetohydroxyacid synthase. Journal of Biochemistry 138: 35–40, 2005. 62. Lee, H., Rustgi, S., Kumar, N., Burke, I., Yenish, J.P, Gil,l K.S., von Wettstein, D. and Ullrich, S.E. Single nucleotide mutation in the barley acetohydroxyacid synthase (AHAS) gene confers resistance to imidazolinon herbicides. PNAS 108: 8909 – 8913, 2011. 63. Lee, K.Y., Townsend, J., Tepperman, J., Black, M., Chui, C.F., Mazur, B., Dunsmuir, P. and Bedbrook, J. The molecular basis of sulfonylurea herbicide resistance in tobacco. The EMBO Journal 7:1241–1248, 1988. 64. Lehrer, S.B. and Bannon, G.A. Risks of allergic reactions to biotech proteins in foods; perception and reality. Allergy 60: 559–564, 2005. 65. Maertens, K.D., Sprague, C.L., Tranel, P.J. and Hines, R.A. Amaranthus hybridus populations resistant to triazine and acetolactate synthase-inhibiting herbicides. Weed research 44: 21–26, 2004. 66. Mapp, C.E., Boschetto, P., Maestrelli, P. and Fabbri, L.M. Occupational asthma. American Journal of Respiratory and Critical Care Medicine 172: 280–305, 2005. 67. Massa, D., Krenz, B. and Gerhards, E. Target-site resistance to ALS-inhibiting herbicides in Apera spica-venti populations is conferred by documented and previously unknown mutations. Weed Research 51: 294 – 303, 2011. 68. Mathesius, C.A., Barnett, J.F., Cressman, R.F., Ding, J., Carpenter, C., Ladics, G.S., Schmist, J., Layton, R.J., Zhang, J.X.Q., Appenzeller, L.M., Carlson, G., Ballou, S. and Delaney, B. Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans. Regulatory Toxicol and Pharm. 55: 309 – 320, 2009. 69. Mazur, B.J., Hui, C-F. and Smith, J.K. Isolation and characterization of plant gene coding for acetolactate synthase, the target enzyme for two classes of herbicides. Plant Physiology 85: 1110–1117, 1987. 70. McBride, J.S., Altman, D.G., Klein, M. and White, W. Green tobacco sickness. Tobacco control 7: 294–298, 1998. 71. McGovern, T.W. and Barkley, T.M. Botanical dermatology in “the electronic textbook of Dermatology” (Drugge R, Ed.) http://www.telemedicine.org/stamford.htm 72. McLachlan, A., Cullis, P.G. and Cornell, H.J. The use of extended amino acid motifs for focusing on toxic peptides in celiac disease. Journal of Biochemistry, Molecular Biology and Biophysics 6: 319–224, 2002. 73. McNaughton, J., Roberts, M., Rice, D., Smith, B., Hinds, M., Delaney, B., Iiams, C. and Sauber, T. Nutritional equivalency evaluation of transgenic maize grain from event DP- 098140-6 and transgenic soybeans containing event DP-356043-5: laying hen performance and egg quality measures. Poultry Science 90: 377 – 389, 2011a Copies of literature review

74. McNaughton, J., Roberts, M., Rice, D., Smith, B., Hinds, M., Delaney, B., Iiams, C. and Sauber, T. Comparison of broiler performance and carcass yields when fed transgenic maize grain containing event DP-098140-6 and processed fractions from transgenic soybeans containing event DP-356043-5. Poultry Science 90: 1701 – 1711, 2011b. 75. McSheffrey, S.A, McHughen, A. and Devine, D. Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum). Theoretical and Applied Genetics 84: 481-486, 1992. 76. Meija, L., Jacobs, C.M., Utterback, P.L., Parsons, C.M., Rice, D., Sanders, C., Smith, B., Iiams, C. and Sauber, T. Evaluation of the nutritional equivalency of soybean meal with the genetically modified trait DP-305423-1 when fed to laying hens. Poultry Science 89: 2634 – 2639, 2010 77. Miesen, W.M.A.J., van der Heide, S., Kerstjens, H.A.M., Dubois, A.E.J. and de Monchy, J.G.R. Occupational asthma due to IgE mediated allergy to the flower Molucella Laevis (bells of Ireland) Occupational and Environmental Medicine 60: 701–703, 2003. 78. Mills, E.N.C., Jenkins, J.A., Alcocer,M.J.C. and Sewry, P.R. Structural, biological, and evolutionary relationships of plant food lergens sensitizing via the gastrointestinal tract. Critical Review of Food Science and Nutrition 44: 379–407, 2004. 79. Mitchell, J. and Rook, A. Botanical dermatology. Available on-line at http://bodd.cf.ac.uk/index.html, 2006. 80. Mitterman, I., Swoboda, I., Person, E., Eller, N., Kraft, D., Valenta, R. and Heberle-Bors, E. Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profiling expression during pollen maturation. Plant Molecular Biology 27: 137– 146, 1995. 81. Monso, E., Magarolas, R., Radon, K., Danuser, B., Iversen, M., Weber, C., Opravil, U., Donham, K.J. and Nowak, D. Respiratory symptoms of obstructive lung disipase in European crop farmers. Journal of Respiratory and Critical Care Medicine 162: 1246-1250, 2000. 82. Monso, E., Magarolas, R., Badorrey, I., Radon, K., Nowak, D. and Morera, J. Occupational asthma in greenhouse flower and ornamental plant growers. American Journal of Respiratory and Critical Care Medicine 165: 954–960, 2002. 83. Mourad, G., Williams, D. and King, J. A double mutation allele, csr1-4, of Arabidopsis thaliana encodes an acetolactate synthase with altered kinetics. Planta 196: 64–68, 1995. 84. Nelson, D. R and Duxbury, T. The distribution of acetohydroxyacid synthase in soil bacteria. Antonie van Leeuwenhoek 93: 123 – 132, 2008. 85. Newhouse, K., Singh, B., Shaner, D. and Stidham, M. Mutations in corn (Zea mays L.) conferring resistance to imidazolinone herbicides. Theoretical and Applied Genetics 83: 65– 70, 1991. 86. Nielsen, K. and Boston, R.S. Ribosome-inactivating proteins; a plant perspective. Annual Review of Plant Physiology and Plant Molecular Biology 52: 785–816, 2001. 87. OGTR. Risk assessment and risk management plan. Application for license for dealings involving an intentional release into the environment. DIR027/2002. Field trial of pineapple plants modified to control flowering, 2003. 88. Ortega, N., Quiralte, J., Blanco, C., Castillo, R., Alvarez, M..J and Carrillo ,T. Tobacco allergy; demonstration of cross-reactivity with other members of Solanaceae family and mugwort pollen. Annals of Allergy, Asthma and Immunology 82: 194–197, 1999. 89. Ouellet, T., Rutledge, R.G. and Miki, B.L. Members of the acetohydoxyacid synthase multigene family of Brassica napus have divergent patterns of expression. Plant Journal 2: 321–330, 1992. 90. Park, K.W. and Mallory-Smith, C.A. Physiological and molecular basis for ALS inhibitor resistance in Bromus tectorum biotypes. Weed research 44: 71–77, 2004. Copies of literature review

91. Pirrila, P., Kanerva, L., Alanko, K., Estlander, T., Keskinen, H., Pajari-Backas, M. and Tuppurainen, M. Occupational IgE-mediated asthma, rhinoconjunctivitis, and contact urticaria caused by Easter lily (Lilium longiflorum) and tulip. Allergy 54: 273–277, 1999. 92. Radauer, C. and Breiteneder, H. Pollen allergens are restricted to few protein families and show distinct patterns of species distribution. Journal of Allergy and Clinical Immunology 117: 141-147, 2006. 93. Rademaker, M. Occupational contact dermatitis to Hydrangea. Australian Journal of Dermatology 44: 220–221, 2003. 94. Ray, T.B. Site of action of chlorsulfuron. Plant Physiology 75: 827–831, 1984. 95. Riaz, T., Hor, L.H., Krishnan, A., Tang, F. and Li, K-B., WebAllergen: web server for predicting allergenic proteins. Bioinformatics 21: 2570–2571, 2005. 96. Romagnoli, S., Ugolini, R., Fogolari, F., Schaller, G., Urech, K., Giannattasio, M., Ragona, L. and Molinari, H. NMR structural determination of viscotoxin A3 form Viscum album L. Biochemistry Journal 350: 569–577, 2000. 97. Rudiger, H. and Gabius, H-J. Plant lectins; occurrence, biochemistry, functions and applications. Glycocojugate Journal 18: 589–613, 2001. 98. Schnell, J., Labbe, H., Kovinich, N., Manabe, Y. and Miuki, B. Comparability of imazapyr- resistant Arabidopsis created by transgenesis and mutagenesis. Transgenic Research 21: 1255 – 1264, 2012 99. Schloss, J.V., Ciskanick, L.M. and Van Dyk, D.E. Origin of the herbicide binding site of acetolactate synthase. Nature 331: 360–362, 1988. 100. Shaner, D.L., Anderseon, P.C. and Stidham, M.A. Imidazolinones. Plant Physiology 76: 545–546, 1984. 101. Shewry, P.R., Beaudoin, F., Jenkins, J., Griffiths-Jones, S. and Mills, E.N.C. Plant protein families and their relationships to food allergy. Biochemical Society Transactions 30: 906–910, 2002. 102. Sibony, M., Michel, A., Haas, H.U., Rubin, B. and Hurle, K. Sulfometuron-resistant Amaranthus retroflexus; cross-resistance and molecular basis for resistance to acetolactate synthase-inhibiting herbicides. Weed Research 41: 509–522, 2001. 103. Singh, B.K. and Sander, D.L. Biosynthesis of branched chain amino acids: from test tube to field. Plant Cell 7: 935–944, 1995. 104. Southan, M.D. and Copeland, L. Physical and kinetic properties of acetohydroxyacid synthase from wheat leaves. Plant Physiology 98: 824–832, 1996. 105. Stadler, M.B. and Stadler, B.M. Allergenicity prediction by protein sequence. FASEB Journal 17: 1141–1143, 2003. 106. Subramanian, M.V., Hung, H-Y., Dias, J.M., Miner, V.W., Butler, J.H. and Jachetta, J.L. Properties of mutant acetolactate synthase resistant to triazoloopyrimidine sulfonanilide. Plant Physiology 94: 239–244, 1990. 107. Tan, S., Evans, R.E., Dahmer, M.L., Singh, B.K. and Shaner, D.L. Imidazolinone- tolerant crops: history, current status and future. Pest Management Science 61: 246–257, 2005. 108. Tranel, P.J., Jiang, W., Patzoldt, W.L. and Wright, T.R. Intraspecific variability of the acetolactate synthase gene. Weed Science 52: 236–241, 2004. 109. USFDA. Biotechnology consultation note to the file BNF No. 000030. Cotton line 19-51a. 1996. 110. USFDA. Biotechnology consultation note to the file BNF No. 000050. Sulfonylurea tolerant linseed flax. 1998. 111. van Ree, R. Clinical importance of non-specific lipid transfer proteins as food allergens. Biochemical Society Transactions 30: 910–913 2002. 112. van der Zee, J.S., Koos, A., de Jager, S.N., Kuipers, B.F. and Stapel, S.O. Outbreak of occupational allergic asthma in a Stephanotis floribunda nursery. Journal of Allergy and Clinical Immunology 103: 950-952, 1999. Copies of literature review

113. Vidal, C. and Polo, F. Occupational allergy caused by Dianthus caryophyllus, Gypsophila panicilata and Lilium longiflorum. Allergy 53: 995–998, 1998. 114. Wagner, S. and Breiteneder, H. The latex-fruit syndrome. Biochemical Society Transactions 30: 935–940, 2002. 115. White, AD, Graham, MA and Owen, MDK. Isolation of acetolactate synthase homologs in common sunflower. Weed Science 51: 845–853, 2003. 116. Wopfner, N, Gadermaier, G, Egger, M, Asero, R, Ebner, C, Jahn-Schmid, B and Ferreira, F. The spectrum of allergens in ragweed and mugwort pollen. International Archives of Allergy and Immunology 138: 337–346, 2005. 117. Yoon, T-Y, Chung, S-M,Chang, S-I,Yoon, M-Y,Hahn, T-R and Choi, J-D. Roles of lysine 219 and 255 residue in tobacco acetolactate synthase. Biochemical and Biophysical Research Communications 293: 433–439, 2002. 118. Yu, Q, Han, H, Vila-Aiub, MM and Powles, SB. AHAS herbicide resistance endowing mutations; effect on AHAS functionality and plant growth. Journal Experimental Botany 61: 3925 – 3934, 2010. Copies of literature reviews

AUGUST 2014 Up-to-date literature search on information relevant for the safety of GM carnation lines IFD- 26407-2 and IFD-25958-3 to humans

CONTENTS

1. Methodology 2. Baseline knowledge; non-GM carnation 2.1. Ornamental use…………………………………………..……4 2.2. Non-ornamental use………………………………...…………4 2.3. Allergenicity of carnation……………………………………..4 2.4. Toxicity of carnation………………………………..…………5 3. Characterisation of inserted genes 3.1. Flavonoid 3’5’- hydroxylase………………………….……….6 3.2. SuRB (acetolactate synthase)……………………………...…..7 3.3. DFR………………………………………………..…………..7 3.4. Cytb5…………………………………………………………..7 4. Characterisation of expressed proteins 4.1. Flavonoid 3’5’- hydroxylase……………………………….….8 4.2. SuRB (acetolactate synthase)………………………………….8 4.3. DFR…………………………………………………...…...…..9 4.4. Cytb5…………………………………………………...….…..9 5. Anthocyanins 5.1. Distribution in plants………………………………………….10 5.2. Anthocyanins in human diet…………………………………..10 5.3. Effects on health………………………………………...…….10 5.4. Toxicology and allergenicity……………………………….…11 6. Delphinidin 6.1. Distribution in ornamental plants……………………………..12 6.2. Distribution in foods…………………………………………..12 6.2.1. Estimated consumption…………………………………....…..13 6.2.2. Metabolism…………………………………………...…...…..13 6.3. Toxicology and allergenicity……………………………….….13 6.4. Effects on health…………………………………………….…14 6.4.1. Anti-cancer effects………………………………………...…..14 6.4.2. Cardiovascular effects……………………………………..…..15 6.4.3. Other effects……………………………………………...……17 7. Potential hazards associated with use of transgenic, colour-modified, carnation 7.1. Use as an ornamental product…………………………………17 7.2. Potential use in perfume industry………………………...... …17 7.3. Potential use as food additive……………………………...….17 7.4. Potential use as food or animal feed………………………..…18 7.5. Production of delphinidin in other plant species…………...…18 7.5.1. Anthocyanin production in transgenic tomato……………..…18 8. Conclusions………………………………………………………………19

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1. Methodology

Dossiers C/NL/09/01 and C/NL/09/02 were compiled at the end of 2008 and included a literature review carried out at that time. Independently of the request from the working group the literature has been continuously reviewed since 2008 as part of a) an annual monitoring process for GM carnation varieties already released in the EU b) marketing applications for transgenic carnation varieties SHD-27531-4 and FLO-40685-2 (lodged in the EU in 2013).

In this document the literature is reviewed by subject, in accordance with the parts of dossiers C/NL/09/01 and C/NL/09/02 which included information relevant to an assessment of possible impacts of GM carnation on human health. For cross reference to the literature review in this document a summary table is provided in Table 1.

Table 1. Cross reference between update of literature and relevant sections in dossiers C/NL/09/01 and C/NL/09/02 Subject Dossier Dossier Relevant C/NL/09/01 C/NL/09/02 part of (IFD-25958-3) (IFD-26407-2) section A of Section, page Section, page this number number document

Baseline knowledge of toxicity of non-GM B2, page 1 B2, page 1 2.4 carnation Overview of plants containing delphinidin- B2, page 3 B2, page 3 6.1 related anthocyanins Theoretical potential for any change in toxicity B2, page 3 B2, page 3 3 or allergenicity in the GMHP due to inserted DNA Theoretical potential for any change in toxicity B2, page 3 B2, page 3 4 or allergenicity in the GMHP due to expressed proteins Baseline knowledge of toxicity of delphinidin- B2, page 3 B2, page 3 6.3 related anthocyanins Potential toxicity and allergenicity of the Attachment Attachment 4.2 acetohydroxyacid synthase (ALS) protein B1 B1 Traditional uses of carnation by humans Not included Not included 2.1 and 2.2 Possibility of use of imported flowers in Not included Not included 7.2 and 7.3 perfume or additive industry Possibility of consumption of imported flowers Not included Not included 7.4 as a food

The literature review in section A was carried out using PubMed, Web of science (ISI), SCOPUS (Elsevier), Science direct, Toxline, Science citation index, MEDLINE (proQuest) and Google scholar databases. This document includes new citations, not included in C/NL/09/01 and C/NL/09/02, as well as key references from those two dossiers.

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2. Baseline knowledge; non-GM carnation

2.1. Ornamental use

Double-flowered varieties of the carnation have been grown as an ornamental plant in Europe since the 15th century (Harvey, 1978; OGTR, 2006). Since then the carnation has been commercialised in all parts of the world and is one of the most common cut-flower ornamental crops. Billions of flowers are grown and sold each year. Details of ornamental use of carnation were provided in attachment B3 of dossiers C/NL/09/01 and C/NL/09/02.

2.2. Non-ornamental use

Traditional non-ornamental use of carnation by humans was not reviewed in carnation dossiers C/NL/09/01 and C/NL/09/02.

Traditional use of Dianthus caryophyllus for medicinal and nutritional use has recently been summarised by Lim (2014). Aside from the primary use of the plant and flowers for decorative purposes, there are reports of the use of carnation in traditional cosmetics (Pieroni et al., 2004) and medicine (Blanco et al., 1999; Bussmann et al., 2011; Mlcek and Rop, 2011; Muthanna and Al-Bayati, 2009; OGTR, 2006). Adams et al. (2012) has described carnation as a plant ingredient in ancient herbal remedies. Carnation petals are one of the ingredients in the alcoholic drink green chartreuse (OGTR, 2006) and in some Turkish baths (Ugulu, 2012). Where carnation has been used as a traditional medicine, crude extracts or dried plant material are employed (Lim, 2014), rather than compounds purified from these extracts. Martineti et al. (2010) showed the compound kaempferide triglycoside, extracted and purified from a commercial cultivar of carnation, could inhibit the growth of human colon cancer cells (Martineti et al., 2010).

Dianthus species other than carnation are also used in traditional medicine (Gou et al., 2011; Lamula and Ashafa, 2014; López-Expósitol et al., 2011) and recent reports in which extracts from other Dianthus species are reported to have positive benefits on human health have been made by Ding et al. (2013), Gevrenova et al. (2014), Shin et al. (2013) and Yu et al. (2012).

We are not aware of any reports of carnation being used as food (also see section 7.4 for further discussion). Neither carnation nor Dianthus is listed on; • The catalogue of novel food products in the EU (EU, online, a) • Novel foods and novel food ingredients that may be placed on the market in the EU pursuant to Regulation (EC) No. 258/97 (EU, online, b)

2.3. Allergenicity of carnation

Flowers are not listed with tree nuts, legumes, fruit, shellfish, eggs, cows milk etc. as a common source of food allergy and carnation pollen cannot be a source of hay fever type allergies as the pollen is not wind-borne. Suntory Ltd. has close relationships with carnation growers in South America and none of these contacts have experienced allergy problems among their staff (refer to section B of this document).

In dossiers C/NL/09/01 and C/NL/09/02 we noted allergic reactions to carnation to be rare. At that time we found in the literature a report of allergenicity in Spanish flower farm workers exposed to carnation (Sánchez-Fernández et al., 2004; Sanchez-Guerrero et al., 1999). Later studies indicated that spider mites associated with the carnation were the cause of the response (Cistero-Bahima et al., 2000; 3

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Navarro et al., 2001; Orta et al., 1998; Vidal and Polo, 1999). Baur (2009) has included carnation and its associated mites in a comprehensive list of potential allergens and irritants in the workplace.

A report of respiratory allergy to carnation has recently been published (Brinia et al., 2013). The report claims to be the first case of a person with no occupational exposure to carnation, though the person regularly handled carnation on social occasions. The responsible allergen was shown to be heat stable, found in all parts of the plant and also found in gypsophila (from the same taxonomic family as carnation). The clinicians assumed the allergen was inhaled in plant material. There are three reports in the literature of mild contact dermatitis associated with handling of carnation (Lamminpaa et al., 1996; Stefanaki and Pisios, 2008; van Grutten, 1980). We were unaware of these reports previously and they were not included in dossiers C/NL/09/01 and C/NL/09/02. Lamminpaa et al. (1996) including carnation extract in patch tests on gardeners and florists using a range of ornamental plants and found, relative to other plants, minor eczema reactions in 2 patients.

No allergic effects were noted by Lim (2014) in a recent review of the nutritional and medicinal properties of carnation.

2.4. Toxicity of carnation

In dossiers C/NL/09/01 and C/NL/09/02 an evaluation of the toxicity of non-GM carnation was made using a series of web sites, some of which have now been terminated. An up-to-date summary of evaluations of the toxicity to humans of non-GM Dianthus/carnation is provided in Table 2.

Table 2. Evaluations of the potential toxicity of carnation online Citation Plant Summary of toxicity NCSU, online Dianthus Causes only low toxicity if eaten. Skin irritation species minor, or lasting only for a few minutes. USFDA, online Carnation Citations on allergenicity and dermatitis are linked to a search on carnation UA, online Carnation Regarded as non-poisonous CPPIS, online Carnation Carnation is not listed as a poisonous plant.

The information supports the conclusion made in dossiers C/NL/09/01 and C/NL/09/02 that at worst non-GMO can be considered to have mild toxicity, but it is generally regarded as non-poisonous and safe. Carnation is not a food and there is no toxicology or nutritional data available for the recipient organism. In Peru, carnation is listed as a traditional medicine and Bussmann et al. (2011) assessed a range of medicinal medicines from that country for toxicity, including Dianthus caryophyllus. Aqueous and ethanol extracts of carnation were shown to be non – toxic in a brine shrimp lethality assay. No toxic effects on humans were noted by Lim (2014) in a recent review of the nutritional and medicinal properties of carnation.

Galeotti et al. (2008) demonstrated that flavonoids isolated from Dianthus caryophyllus inhibited growth of the fungal pathogen Fusarium oxysporum.

Extracts of Dianthus caryophyllus seed were shown to have antiviral activity (against HSV-1 and HAV-27) in a plaque infectivity count assay (Barakate et al., 2010). Kimbaris et al. (2012) provided evidence for inhibitory effect of Dianthus caryophyllus oil extract on the West Nile disease vector Culex pipiens.

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Commercial flower growers routinely use chemicals for pest and pathogen control (Brouwer et al., 1992). Bolognesi et al. (2002) found an increase in micronuclei in Italian workers handling flowers, including carnation, and attributed this to pesticide exposure.

3. Characterisation of inserted genes

In dossiers C/NL/09/01 and C/NL/09/02 it was stated there was no reason to believe the DNA per se could be a reasonable cause of any increase in toxicity or allergenicity. An up-to-date BLASTn bioinformatic analysis was carried out in November 2013, using both the inserted DNA sequences and all theoretical ORFs (from insert and flanking sequence) as query sequences. The analysis was carried out for both IFD-26407-2 and IFD-25958-3. The results, provided to the molecular characterisation working group of the GMO panel, concluded; a) No homology to allergen or toxin proteins was identified through bioinformatic analysis of the nucleotide or amino acid of the sequences flanking the inserted DNA in either IFD- 25958-3 or IFD-26407-2. b) No homology to allergen or toxin proteins have been identified through bioinformatic analysis of the ORFs generated from the sequences flanking the inserted DNA in either event. c) The inserted T-DNA components showed no significant homology to known toxin or allergen proteins.

As stated in dossiers C/NL/09/01 and C/NL/09/02 these observations may be expected from the ubiquitous presence of homologs of the inserted genes in nature and the fact they are not known to encode toxin or allergen proteins.

Since C/NL/09/01 and C/NL/09/02 were written, significant advances have been made in understanding the genetics of anthocyanin and pigment biosynthesis in carnation (Ohmiya et al., 2013; Sasaki et al., 2013), including publication of a draft genome sequence (Yagi et al., 2013). The availability of the carnation sequences, supplemented with data about anthocyanin biosynthesis genes from other species (Yamagishi, 2013; Yuan et al., 2013) has enhanced the value of bioinformatic analysis.

Flavonoid 3'5'- hydroxylase and cytochrome b5 are both microsomal proteins and during the literature review we did not found any reports in which these proteins (of plant origin) were isolated and characterised. In such cases, and where there is clearly a history of safe use and dietary and dermatological exposure (given the ubiquitous nature of the proteins in ornamentals and foods used by human), the bioinformatic analysis becomes an important tool in risk assessment (Bushey et al., 2014). This is because, where the protein cannot be isolated, no digestibility or tier II animal feeding testing can be carried out.

3.1. Flavonoid 3'5'- hydroxylase

Since C/NL/09/01 and C/NL/09/02 were written, genes encoding flavonoid 3'5'-hydroxylase have been sequenced and characterised from pea (Moreau et al., 2012), delphinium (Miyagawa et al., 2014), Epimedium sagittatum (Huang et al., 2012), Antirrhinum kelloggii (Ishiguro et al., 2012), Pohlia nutans (Liu et al., 2014), cineraria (Sun et al., 2013) and soybean (Guo and Qiu, 2013). The BLASTn bioinformatic analysis carried out in November 2013 showed the query sequence for the flavonoid 3'5'-hydroxylase petunia and pansy genes used in IFD-25958-3 or IFD-26407-2 had highest homology (measured by lowest E-value) to flavonoid 3' 5'- hydroxylase genes from other species. No significant homology to known toxin or allergen proteins was identified.

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3.2. SuRB (acetolactate synthase)

The BLASTn bioinformatic analysis carried out in November 2013 showed the query sequence for the SuRB sequence used (identical in both IFD-25958-3 and IFD-26407-2) had highest homology to acetolactate synthase genes from other species. No significant homologies to known toxin or allergen proteins were identified.

3.3. DFR

Mori et al. (2014) isolated the dihydroflavonol 4-reductase gene from Agapanthus praecox and found it to be functional when expressed in transgenic petunia under control of the constitutive 35S promoter. The BLASTn bioinformatic analysis carried out in November 2013 showed the query sequences for both the sense and anti-sense dihydroflavonol 4-reductase sequences used in IFD-25958-3 had highest homology to dihydroflavonol 4-reductase genes from other species. No significant homologies to known toxin or allergen proteins were identified.

3.4. Cytb5

The BLASTn bioinformatic analysis carried out in November 2013 showed the query sequence for the Cytb5 sequence used (from IFD-26407-2) had highest homology to cytochrome b5 genes from other species. No significant homologies to known toxin or allergen proteins were identified.

4. Characterisation of expressed proteins

In dossiers C/NL/09/01 and C/NL/09/02 it was stated the proteins encoded by the inserted genes (table 3) are common plant proteins and are not toxic or allergenic, nor homologous to known toxic or allergenic proteins

Table 3. Introduced proteins expressed in IFD-25958-3 and IFD-26407-2 IFD-25958-3 IFD-26407-2 Gene Protein Gene Protein SuRB ALS (acetolactate synthase) SuRB ALS (acetolactate synthase) F3'5'H F3'5'H (flavonoid 3'5'- F3'5'H F3'5'H (flavonoid 3'5'- hydroxylase) hydroxylase) Sense and DFR (dihydroflavonol 4- Cytb5 Cytb5 (cytochrome b5) antisense DFR reductase)

An up-to-date BLASTp bioinformatic analysis was carried out in October 2013, using the inserted protein sequences as query sequences. The analysis was carried out for both IFD-26407-2 and IFD- 25958-3. Though significant alignments with low E-values were found in blastp analysis no biologically significant similarity to known toxins or allergen proteins was identified. Details of the analysis were provided to the molecular characterisation working group in November 2013.

None of the inserted proteins have the characteristics of toxin proteins (reviewed in Wu and Sun, 2012) and as the inserted genes are common plant proteins, they would be degraded in the same way as similar proteins found in common food (refer to parts 5 and 6). The amino acids produced after digestion will be excreted or metabolized in the same way as those from other plant proteins.

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4.1. Flavonoid 3'5'- hydroxylase

The flavonoid 3'5'- hydroxylase protein is found in all plant foods producing the anthocyanin delphinidin. This includes several raw foods containing high levels of delphinidin, normally consumed and handled by humans, and known to be safe (refer to part 6).

4.2. SuRB (acetolactate synthase)

The SuRB gene is a mutation of an enzyme found in all plants and as such, the amino acid sequence and protein is virtually the same as that of the enzyme found in all raw plant foods. A detailed review of the potential toxicity and allergenicity of the SuRB was given in Attachment B1 of dossiers C/NL/09/01 and C/NL/09/02. That review has been updated for this document to include recent safety assessments using GM plants.

To our knowledge, it remains the case that no food crop variety has been commercialized in which the SuRB gene mutation used in carnation events IFD-26407-2 and IFD-25958-3 has been used in the transformation vector. However, the gm-hra gene, a modified soybean ALS gene, has mutations analogous to those of SuRB, and transgenic plants containing gm-hra have been studied in several animal feeding studies. The results from these studies are summarised in table 4. The studies have also been included in a recent review of the validity of using animal feeding studies for determining the safety of GM crops (Bartholomaeus et al., 2013).

Table 4. Animal feeding studies in which seed and extracts from transgenic plants containing modified ALS genes have been compared to appropriate non-transgenic control lines Reference Test Source Characterization Conclusion ALS gene or animal protein Appenzeller Rat Soybean Body weight, clinical No biologically gm-hra et al., 2008 signs, mortality, sensory relevant adverse effects response, clinical pathology, organ weights, gross pathology, microscopic pathology Appenzeller Rat Maize Body weight, No biologically zm-hra et al., 2009 Ophthalmology, relevant adverse effects Neurobehavioral evaluation, clinical pathology, gross and anatomical pathology Chukwudbe Rat Soybean Body weight, organ No substantial csr 1-2 et al., 2012 weights, Clinical alteration to pathology, gross necropsy composition of and histopathology soybeans. No adverse effects on nutritional or safety status. Delaney et Rat Soybean Body weight, clinical No biologically gm-hra al., 2008 signs, mortality, toxicity, relevant adverse effects sensory response, clinical pathology, organ weights, gross pathology, clinical pathology Mathesius Mice Purified Body weight and lesions at No significant Purified et al., 2009 protein necropsy differences. protein from gm-hra gene

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Reference Test Source Characterization Conclusion ALS gene or animal protein administered at 463 mg/kg body weight Mathesius Mice Purified Body weight, clinical and No biologically Purified et al., 2009 protein ophthalmological relevant adverse effects protein from observations, motor gm-hra gene activity, clinical chemistry administered response, histopathological in food at observations 1000 mg/kg body weight McNaughton Chicken Maize and Weight and egg No significant zm-hra, gm- et al., 2011a Soybean production, cracking differences. hra McNaughton Chicken Maize and Weight gain, feed intake, No significant zm-hra, gm- et al., 2011b Soybean carcass yield differences. hra Meija et al., Chicken Soybean Weight and egg production No significant gm-hra 2010 and quality, cracking differences.

As table 4 shows, there was no effect of expression of the ALS gene in the GM plant that could have an effect on human health (Mathesius et al., 2009). ALS has not been identified as an allergen despite the presence of this protein in all plants. A search of the plant food-allergens, aero-allergens and contact allergens in the Faarp protein database of known allergens (FAARP, online) did not list ALS from any source.

Mathesius et al. (2009) isolated and purified the protein from the gm-hra gene (table 4) and showed that the protein was quickly degraded by the digestive enzymes pepsin or pancreatin. The same authors demonstrated that enzymatic activity was lost at temperatures above 50ºC. These characteristics are not usually associated with a protein food allergen.

4.3. DFR

The DFR protein is found in all plant foods containing anthocyanins (please refer to part 5). This includes many raw foods containing high levels of anthocyanin, known to be safe (refer to part 5).

An antisense DFR construct (utilised in the transformation vector used to produce IFD-25958-3) has been used to transform Nelumbo nucifera, but no phenotypically altered plants were obtained (Buathong et al., 2013).

4.4. Cytb5

Compared to the other inserted genes in IFD-26407-2 and IFD-25958-3, cytochrome b5 proteins are more ubiquitous, as they are found in plants, animals, fungi and some bacterial species. It is reasonable to assume some cytochrome b5 protein is part of all human diets. Like flavonoid 3'5'- hydroxylase protein, cytochrome b5 is membrane bound and so may be considered intractable (Bushey et al., 2014).

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5. Anthocyanins

5.1. Distribution in plants

Anthocyanins are present in numerous ornamental and food plants. Recent reviews of anthocyanin biosynthesis and distribution include Anantharaman et al. (2014), Corcoran (2012), Kruger et al. (2014), Martin et al. (2013) and Tsuda (2012).

5.2. Anthocyanins in human diet

A dietary survey by Wu et al. (2006) estimated that the daily intake of anthocyanins in the US population was 12.5 mg/day/person, with cyanidin, delphinidin and malvidin contributing to approximately 45, 21 and 15%, respectively, of total anthocyanin intake. A review of the dietary intake of flavonoids, including anthocyanidins, in other countries suggest the US intake is similar to other countries of the world but may be lower than those where berry consumption is high. For example, Heinonen (2007) has estimated Finns eat approximately 80 mg anthocyanin per day. Anthocyanins are likely to be present in large amounts in some individual diets because of the high concentration of anthocyanins in some food sources. For example, 100g of berries may have approximately 500 mg of anthocyanins, translating to 100 – 300 mg anthocyanin per serve (Corcoran, 2012).

5.3. Effects on health

Major health benefits attributed to anthocyanin consumption include improved cardiovascular health (Kruger et al., 2014; Lin et al., 2014; Quinones et al., 2013; Son et al., 2014; Yamagata et al., 2014), increased resistance to viruses, suppression of cancer cell growth (Anantharaman et al., 2014; Mazzucato et al., 2013; Zhang et al., 2005) possible anti-obesity agents (Shan et al., 2014) and treatment of infection (Broadhurst, 2001; Martin et al., 2013). Anti-diabetic properties have also been reviewed (Sancho and Pastore, 2012). The health properties of anthocyanins are further described in Sterling (2001) Norberto et al. (2013) and Lila (2004).

The anthocyanidin cyanidin was shown by Wang et al. (1999) to be an anti-inflammatory and anti- microbial compound. Mice fed black rice extract (containing primarily cyanidin-based anthocyanins) displayed significant increases in superoxide dismutase (SOD) and catalase (CAT) activity while superoxide anions (O2-) and reactive oxygen species were significantly suppressed, demonstrating the anti-oxidant effect of anthocyanins (Chiang et al., 2006). Anthocyanins have been reported to have positive effects on brain function (Kim et al., 2012b) and act as anti-obesity (Kim et al., 2012a) and anti-inflammatory agents (Zhu et al., 2013). Ogawa et al. (2013, 2014) have demonstrated, using cultures mouse cells, that anthocyanins may confer protection against UV light.

Several reviews summarize the scientific rationale for the positive effects of polyphenols and flavonoids, including anthocyanins, on health (Beecher, 2003; Bueno et al., 2012a; Corcoran, 2012; Heinonen, 2007; Manach et al., 2005; Nichenametla et al., 2006; Tsuda, 2012). Berry phenolics, rich in anthocyanins, have anti-oxidant and anti-microbial effects (Bornsek et al., 2012; Céspedes et al., 2010; Cui et al., 2013; Heinonen, 2007; Li et al., 2012; Miladinovic et al., 2014; Murapa et al., 2012).

Consumption of tomato genetically modified for enhanced anthocyanin accumulation was found to enhance the life span of cancer-susceptible mice (Butelli et al., 2008; Gonzali et al., 2009).

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Over 600 anthocyanins have been identified (Bueno et al., 2012b; Prior and Wu, 2006) but little is known about the effect of the anthocyanidin moieties on uptake rates. The current state of knowledge is reviewed by Fernandes et al., 2014). For flavonoids such as quercetin, conjugated forms are more easily taken up than the aglycone (Williamson et al., 2005) and it is known for other flavonoids that the type of moiety may determine the rate of uptake (Crespy et al., 2003; Felgines et al., 2000; Fernandes et al., 2012; Prior and Wu, 2006).

5.4. Toxicology and allergenicity

Allergic reactions to anthocyanin containing foods have been studied because most people are exposed to at least some anthocyanin in the course of their regular diet. A review states that there are no reports of allergic reaction to either grape skin extract or grape colour extract, both of which are widely used food colourants (Lucas et al., 2001). Allergy to berries, rich in anthocyanins, including delphinidin-based anthocyanins, is extremely rare. An example is in Lingonberry, which primarily produces cyanidin (Matheu et al., 2004) and red currant (Zollner et al., 2000). Eriksson et al. (2004) have studied self-reported food hypersensitivity in northern European Nordic countries, where consumption of berries is typically high (Heinonen, 2007). There was no correlation in that study between foods with high levels of anthocyanins and reported hypersensitivity.

Anthocyanins have a low acute toxicity of ca. 20,000 mg/kg BW in rodents, and a very low order of toxicity (WHO, 2001).

Corcoran (2012) has reviewed the safety of flavonoids to human health and did not identify any potential hazards associated with excess dietary intake of anthocyanins. The intrinsic toxicity of anthocyanins used as food colouring agents is considered to be low by the EU scientific committee for food (EC, 1997).

6. Delphinidin

As outlined in dossiers C/NL/09/01 and C/NL/09/02, the transgenic varieties IFD-26407-2 and IFD- 25958-3 are primarily different to their parental controls because the modified phenotype results in the accumulation of delphinidin-related anthocyanins in flowers. The anthocyanin profiles of the varieties are shown in table 5.

Table 5. Anthocyanidin concentration in IFD-26407-2 and IFD-25958-3 and the parental variety the lines were derived from Anthocyanidin mg/g fresh weight petal anthocyanidin Parent (recipient) IFD-26407-2 IFD-25958-3 variety Delphinidin 0 2.87 0.54 Cyanidin 0.01 0.37 0.10 Pelargonidin 1.06 0.01 0.02 % delphinidin 0 88 81

Because the accumulation of delphinidin is novel in carnation (whereas non-modified carnation produces cyanidin and/or pelargonidin) an assessment of potential effects of delphinidin on human health is a critical part of this literature review.

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6.1. Distribution in ornamental plants

As summarised in dossiers C/NL/09/01 and C/NL/09/02 the levels of delphinidin detected in the transgenic flowers of IFD-26407-2 and IFD-25958-3 are within the range seen in common, widely cultivated ornamental plants (table 6).

Table 6. Delphinidin concentration in common delphinidin containing species. Data has been generated by internal analysis, except for Hibiscus (Puckhaber et al., 2002), tropical water lily (Zhu et al., 2012) and Viola sp. (Zhang et al., 2012) Delphinidin (mg/g Delphinidin as % all FW) anthocyanins Agapanthus 0.12 82 Tropical water lily 0.64 Approx. 70 Brachycome 0.75 83 Cineraria 0.96 71 Delphinium 0.52 98 Viola sp. 1.4 Approx. 40 Dampiera 1.64 100 Hibiscus species 1 - 10 Less than 50 Iris 1.26 100 Rhododendron 0.14 50 Lisianthus 2.8 90 Wisteria 0.39 89

Since dossiers C/NL/09/01 and C/NL/09/02 were compiled delphinidin-related anthocyanins have been extracted and characterised from grape hyacinth (Lou et al., 2014), iris (Wang et al., 2013) Ajuga (Inomata et al., 2013) Hosta (Liu et al., 2013), Veronica persica (Ono et al., 2010) and Heliophila coronopifolia (Saito et al., 2011).

6.2. Distribution in foods

Table 7 provides an estimation of the amount of delphinidin in common food plants.

Table 7. Estimation of delphinidin content in common delphinidin containing foods Mean mg delphinidin per 100g edible portion Blackcurrant, raw 181.11 Black beans 11.98 Strawberry, fresh 0.32 Bilberry 161.93 Cranberry, raw 7.66 Raspberry 0.29 Cowpea 94.6 Bananas 7.39 Red cabbage 0.1 Blueberries, fresh 47.4 Pecan nuts 7.28 Green peas 0.03 Blackcurrant juice 27.8 Red grapes, raw 3.67 Red plum 0.02 Blueberries, frozen 21.59 Red onion, raw 2.28 Apples, raw 0.02 Eggplant, raw 13.76 Sweet potato, purple 0.90 Mango, raw 0.02 (USDA, online)

Delphinidin is present in many other plants consumed by humans, including plants traditionally used for medicinal purposes (Ezuruike and Prieto, 2014). For example, delphinidin is also found in wine (Garcia-Estevez et al., 2013), grape (Srovnalova et al., 2014), Aristotelia chilensis (Chilean blackberry) (Céspedes et al., 2010), some wheat varieties (Trojan et al., 2014), black soybean (Koh et al., 2014) Corema album (León-González et al., 2013) tamarillo (Osorio et al., 2012), Berberis (Ruiz et al., 2013) and Rhodomyrtus tomentose (Cui et al., 2013).

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6.2.1. Estimated consumption

The daily estimated consumption of delphinidin as the anthocyanidin, from commonly consumed fruits and vegetables in the American diet, has been estimated at 2.5 mg per day (Wu et al., 2006) and in France 1.6 mg per day (Perez-Jimenez et al., 2011, with supplement). In France, daily consumption of malvidin (a delphinidin-related anthocyanin) was estimated at 16 mg per adult (Perez-Jimenez et al., 2011). Strawberry, cherry, banana and blueberry are the major contributors to total flavonoid intake in the USA (Chun et al., 2012). The Finnish population eat ca. 80 mg anthocyanin per day, with berries such as blackcurrant and bilberry a primary source of anthocyanin (Heinonen, 2007). Single-dose intake of several hundred milligrams is quite reasonable (Manach et al., 2005). The concentration of delphinidin we have measured in the transgenic flowers of IFD-26407-2 and IFD-25958-3 is comparable to the level in blueberry, as an example, which may have up to 5 mg anthocyanin per g FW, 40% of which is delphinidin-based (Kalt et al., 1999).

6.2.2. Metabolism

In flowers of the transgenic lines the delphinidin likely occurs in the conjugated form. The primary forms are most probably (Fukui et al., 2003); • Delphinidin 3 -(6″-O-4-malyl-glucosyl)-5-glucoside • Delphinidin 3-(6″-O-4-malyl-glucosyl)-5-(6′′′-O-1-malyl-glucoside) • Delphinidin 3,5-diglucoside-6″-O-4, 6′′′-O-1-cyclic-malyl diester

Delphinidin –based anthocyanins are absorbed relatively quickly and though they are primarily not excreted (Wu et al., 2005) they disappear from circulation within 6 – 8 hours (Manach et al., 2005). Delphinidin-related anthocyanins are more susceptible to degradation in saliva than other anthocyanins (Kamonpatana et al., 2012). Glucuronidation and methylation has been demonstrated to be a major metabolic pathway of anthocyanins (Manach et al., 2005) but delphinidin-based anthocyanins are not metabolized in this way in pig (Wu et al., 2005). Anthocyanins move rapidly from the stomach to the brain (Manach et al., 2005). Srovnalova et al. (2014) have established that inclusion of anthocyanins, including delphinidin, in the diet is unlikely to induce expression of major drug-metabolizing enzymes in humans.

6.3. Toxicology and allergenicity

Delphinidin is not known to be a toxic compound, when consumed or when handled. There is no toxicity data in the Merck Index for the aglycone, the mono-glucoside or the 3, 5-diglucoside of delphinidin. As outlined above delphinidin is found in many raw foods (table 7).

While the literature overwhelmingly indicates anthocyanins in general, and delphinidin specifically, are beneficial for human health ( see part 6.4) we are aware of a report in which delphinidin was reported to have an indirect toxic effect, through promotion of tumour cell growth in rat MT-450 mammary carcinoma cells (Thiele et al., 2013).

While there is no literature we are aware of reporting studies of the allergenicity of delphinidin, bilberry extract (rich in delphinidin-based anthocyanins) did not elicit an allergenic reaction (More et al., 2003).

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6.4. Effects on health

A recent review by Patel et al. (2013) provides an overview of the many reported medicinal and pharmacological benefits of delphinidin. Several studies have compared the relative efficacy of the major anthocyanins and have shown that delphinidin-based anthocyanins have greater activity than other anthocyanins (Hou et al., 2005; Khan et al., 2002; Lazze et al., 2006; Lamy et al., 2006, 2007; Noda et al., 2002). In contrast, Guzman et al. (2009) showed delphinidin had a lower antioxidant capacity than other anthocyanins. Like other anthocyanins, delphinidin is an anti-oxidant, the mechanism of which may relate to mobilisation of copper ions (Hanif et al., 2008).

6.4.1. Anti-cancer effects

One of the most widely reported beneficial effects of delphinidin relate to protection from cancer and suppression of growth of cancer cells. Details of some of these reports are summarised in table 8. Delphinidin acts as a broad-spectrum inhibitor of receptor tyrosine kinases in both cell-free and cell systems (Teller et al., 2009).

Table 8. Evidence of anti-cancer effects of delphinidin and delphinidin-based anthocyanins Chemical tested Observed effect In vitro system Citation Delphinidin as pure Inhibition of tumour necrosis JB6 P+ mouse Hwang et al., anthocyanidin factor-a-induced COX-2 epidermal cells 2009 (20 or 40 µM) expression Delphinidin as pure UVB-induced MMP- Cultured human Lim et al., anthocyanidin 1 expression dermal fibroblast 2013 (from 10 µM) Delphinidin as pure Inhibition of proliferation, Cultured human Ozbay and anthocyanidin (50% inhibition blockage of breast cancer Nahta, 2011 at 50 µg/mL) anchorage-independent cells growth and induction of apoptosis Delphinidin as pure Suppression of NF-ĸB Cultured human Yun et al., anthocyanidin pathway, arresting cell cycle colon cancer 2009 (up to 240 µM) cells Delphinidin as pure Induction of autophagic Human hepato- Feng et al., anthocyanidin cellular vacuolization cellular 2010 (from 80 µM) carcinoma cell lines SMMC7721,HC CLM3, andMHCC97L Delphinidin as pure Inhibition of cell growth Human prostate Hafeez anthocyanidin cancer PC3 cells et al., 2008 (up to 100 µmol/L) Delphinidin 3-( p- Angiogenesis inhibitor Human umbilical Matsubara coumaroylrutinoside)-5- - reducing growth rate of vein endothelial et al., 2005 glucoside from eggplant blood vessel cells cells in vitro (10 – 200 µM) Delphinidin as pure Inhibition of epidermal Human breast Afaq et al., anthocyanidin growth factor receptor in cancer cells 2008 (40 µM) cancer cells

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Chemical tested Observed effect In vitro system Citation Delphinidin chloride(IC50 value Inhibition of proliferation and Human hepatoma Yeh and Yen, of 10.8 µM) reduced viability of human cells 2005 hepatoma cells (Hep3B and HepG2) Delphinidin as anthocyanidin Cytotoxicity Cultured human Cvorovic (100 µM) colorectal et al., 2010 carcinoma cells Delphinidin as anthocyanidin Suppression of plasminogen Cultured human Lamy et al., (50 µM) and delphinidin 3- activation, through inhibition glioblastoma cell 2007 glucoside. of expression of both line U-87 urokinase-type plasminogen activator receptor (uPAR) and the low-density lipoprotein receptor-related protein (LRP) and strong inhibition of cell migration Delphinidin chloride, IC50 was Inhibition of glyoxalase I Cultured human Takasawa et 1.9 µM. Tested up to 100 µM (GLO I) activity promyelocytic al., 2010 leukemia HL-60 cells Delphinidin, at a concentration Inhibited vascular endothelial Cultured bovine Nichenametla of 0.01g/L growth factor-induced aortic endothelial et al., 2006 proliferation by blocking cell cells cycle in G0/G1 phase Delphinidin as anthocyanidin Micronuclei in polychromatic Mice feeding Azevedo et al., (10 and 20 mg/kg b.w.) erythrocytes induced by study 2007 cyclophosphamid Anthocyanin extracts (70% of Inhibition of cell growth B16-F10 Bunea et al., which was delphinidin-related metastatic 2013 anthocyanins) higher than 500 melanoma µg/ml. murine cell

Though Kausar et al. (2012) have shown the anti-cancer effect of delphinidin is lower than the synergistic effect with other anthocyanins, Fernandes et al. (2013) has demonstrated that the methylation of delphinidin had no effect in inhibiting growth of three human cancer cell lines in vitro.

6.4.2. Cardiovascular effects

The second most widely reported beneficial effects of delphinidin relate to inhibition of physiological processes that may contribute to cardiovascular disease. Details of some of these reports are summarised in table 9. The effects of delphinidin on cardiovascular disease have been reviewed by Dayoub et al. (2013).

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Table 9. Evidence of positive effects of delphinidin and delphinidin-based anthocyanins on cardio-vascular health Chemical tested Observed effect In vitro system Health effect Citation Delphinidin 3- Suppression of oxidized Human umbilical Improved Jin et al., glucoside (from 10 low-density lipoprotein vein endothelial cardio- 2013 µM) (oxLDL)-induced cell cells vascular proliferation inhibition protection and apoptosis Delphinidin as pure Inhibition of Isolated rat heart Cardio- Scarabelli et anthocyanidin transcription 1 (STAT1) protection al., 2009 (10 µM) activation Delphinidin 3- Inhibition of platelet Mice and human Improved Yang et al., glucoside (up to 50 aggregation derived platelet cardio- 2012 µM) aggregation vascular assay protection Delphinidin 3- Neutralize oxidative Cultured porcine Improved Xie et al., glucoside (up to 150 stress induced by aorticendothelial cardio- 2012 µM) oxidized low-density cells vascular lipoprotein protection Delphinidin 3-O- Inhibition of In vitro Anti- Ojeda et al., sambubioside angiotension I enzymatic assay hypertension 2010 ( 2mg/mL) converting enzyme Delphinidin 3-( p- Angiogenesis inhibitor Human umbilical Anti-cancer Matsubara coumaroylrutinoside) - reducing growth rate vein endothelial and et al., 2005 -5-glucoside from of blood vessel cells cells in vitro Improved eggplant cardio- (10 – 200 µM) vascular protection Delphinidin as pure Suppression of Cultured human Beneficial Lazze et al., anthocyanidin excretion of the umbilical vein effects 2006 (100 µM) vasoconstrictor endothelial cells on the endothelin and cardiovascular stimulation of the system. vasodilator nitric oxide (NO) Delphinidin as Inhibition of expression Lipopolysacchari Anti- Hou et al., anthocyanidin of cyclooxygenase-2 de (LPS)- inflammatory 2005 (COX-2) blocking activated murine nitrogen-activated macrophage protein kinase pathways RAW264 cells Delphinidin as Reduced loss of Cultured human Cardiovascular Chen et al., anthocyanidin (up to viability of low-density umbilical vein – protection 2010 50 µmol/L) lipoprotein as a result of endothelial cells oxidation Delphinidin as 4-fold inhibition of Cultured bovine Cardiovascular Khan et al., anthocyanidin (30 endothelin-1 synthesis aortic endothelial – protection 2002 µM) cells against atherosclerosis Delphinidin chloride Reduced apoptosis Cultured bovine Reduction in Martin et al., (10mg/l) elicited by actinomycin aortic endothelial cardiovascular 2003 D cells disease 15

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Chemical tested Observed effect In vitro system Health effect Citation

Delphinidin as Complete inhibition of Rat brain Anti-oxidant Noda et al., anthocyanidin H2O2-induced lipid homogenate activity 2002 (10µM) peroxidation

6.4.3. Other effects

Other benefits of delphinidin on human health have been reported; • Chamcheu et al. (2013) found that delphinidin induced human epidermal cell differentiation in vitro and suggested the compound could be used as a useful agent to treat dermatoses. • Delphinidin has an anti-inflammatory effect in cultured chondrocytes (Haseeb et al., 2012, 2013) in human rheumatoid arthritis synovial cell lines (Seong et al., 2011) and with α- glucosidase and α –amylase enzyme assays (Johnson et al., 2013). • Delphinidin can increase the immune response by stimulating cytokine production in T cells (Jara et al., 2014). • Moriwaki et al. (2014) showed delphinidin to be the active ingredient in the anti- osteoporotic bone resorption response in mice, suggesting a possible role for delphinidin in treating osteoporosis. • Anthocyanins such as delphinidin-3-glucoside (Dp3G) and cyanidin-3-glucoside (Cy3G) were able to reduce cytochrome c directly and rapidly, whereas pelargonidin-3-glucoside (Pg3G), malvidin-3-glucoside (Mv3G) and peonidin-3-glucoside (Pn3G) had relatively low cytochrome c reducing activities (Skemiene et al., 2013). • Tanaka et al. (2013) demonstrated the protective effects of maqui berry (Aristotelia chilensis) extract on cell death in light-induced murine photoreceptor cells from mouse. The report showed the same effects were observed using pure delphinidin glucosides.

7. Potential hazards associated with use of transgenic, colour-modified, carnation

7.1. Use as an ornamental product

Please refer to section B of this document.

7.2. Potential use in perfume industry

In dossiers C/NL/09/01 and C/NL/09/02 reasons were provided why we felt transgenic carnation flowers would not be used in the perfume industry. To date, this has been the case, for these reasons; • Though available as an anti-fungal and insect repellent, carnation oil is not an important source of essential oils for the fragrance industry (CBI, 2009; Lubbe and Verpoorte, 2011) or in cosmetics (Aburjai and Natsheh, 2003) though it is used in some perfumes (OGTR, 2006). • Essential oil is present in small amounts in petals of the carnation. According to OGTR (2006) about 500kg of flowers are required to produce 100g of oil. • Other varieties of carnation would be equally as useful as a source of essential oils.

7.3. Potential use as food additive

In dossiers C/NL/09/01 and C/NL/09/02 reasons were provided why we felt transgenic carnation flowers would not be used in the flavour industries. To date, this has been the case, which is consistent with the fact that carnation is not a traditional source of flavour additives for the food 16

Copies of literature reviews industry (CBI, 2010) apart from to flavour wine (OGTR, 2006). The database of food additives authorized in the EU (EU, online, c) does not list any Dianthus ingredient. While the transgenic carnation produces delphinidin and that is unique for this species, there are other natural sources of delphinidin for use as a food colorant. The main one of these is enocianina, prepared by the aqueous extraction of fresh, deseeded marc (grape residue after pressing for grape juice or wine) or fruit juice (grape, cranberry, chokeberry or elderberry). These food sources are much less expensive than flowers, have a higher concentration of available anthocyanin and are more suitable because, generally, anthocyanins are unsuitable as colorants due to instability in alkaline pH and high temperatures (Nitteranon et al., 2014).

7.4. Potential use as food or animal feed

In dossiers C/NL/09/01 and C/NL/09/02 reasons were provided for a case that imported carnation flowers would not enter the food chain; • It would make no economic sense to purchase flowers for large scale use as an animal feed. Though carnation waste has been assessed for use as animal feed in areas where there is significant production, such as Spain and Turkey, concerns have been raised about the practice because of the possible contamination of the feed with agricultural chemicals, such as insecticides (Ceron et al., 1996; Yuksel et al., 2010). • The flowers are likely to be purchased for the use they are intended, not for consumption as a food. • The flowers are intended for home and display use, and once distributed will be widely spread, in small numbers. • Used flowers are likely, as a matter of convenience, to be disposed of like other flowers used and not fed to animals, such as pets. Experience of cultivation and distribution of other GM varieties of carnation, also modified to accumulate delphinidin-related anthocyanins, suggest these arguments are true. After 14 years of cultivation and sale we are not aware of any systematic consumption of the GM carnation as a food.

Though not consumed as a major component of the human diet, edible flowers of some species have long been consumed as an accompaniment to food or as a natural medicine (Mlcek and Rop, 2011; Rop et al., 2012). Included in this list of traditionally used flowers is the carnation (Bussmann et al., 2011; Lim, 2014; Mlcek and Rop, 2011; OGTR, 2006). There is therefore a slight possibility that some consumers may decide to garnish foods with petals from IFD-26407-2 and IFD- 25958-3. In the event that this did occur, we do not believe the transgenic carnation poses any health risk and given the established health benefits of delphinidin consumption (part 6) may even have a positive effect. It is reasonable to expect just a few petals would be used as a garnish, which would be comparable to a very small portion of a fruit containing significant amounts of delphinidin.

7.5. Production of delphinidin in other plant species

Since dossiers C/NL/09/01 and C/NL/09/02 were compiled genetic modification for flower colour, resulting in the accumulation of delphinidin-related anthocyanins, has been achieved in petunia (Ishiguro et al., 2012; Qi et al., 2013), tobacco (Sun et al., 2013) and chrysanthemum (Brugliera et al., 2013; Noda et al., 2013). These transgenic lines have not been commercialised and no trial data or biosafety assessment has been published. Transgenic rose carrying a flavonoid 3’5’-hydroxylase and producing delphinidin has been commercialised but the biosafety experiments published have not looked for possible impacts on human health (Nakamura et al., 2011). Further details of recent advances in colour modification are provided in Sasaki and Nakayama (2014).

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7.5.1. Anthocyanin production in transgenic tomato

Since dossiers C/NL/09/01 and C/NL/09/02 were written, reviews of development of transgenic food plants with altered anthocyanin profiles have appeared (Dixon et al., 2013; Zhang et al., 2014). To our knowledge, biosafety experiments have only been carried out in transgenic tomato, engineered to over-produce anthocyanins (Gonzali et al., 2009; Maligeppagol et al., 2013). The modification was the result of transcription factor modification, not the insertion of the genes that have been used to modify IFD-26407-2 and IFD-25958-3. However, some of the anthocyanins accumulated were delphinidin-related (Butelli et al., 2008).

Consumption of tomato genetically modified for enhanced anthocyanin accumulation was found to enhance the life span of cancer-susceptible mice (Butelli et al., 2008; Gonzali et al., 2009). The antioxidant activity of transgenic fruit was 2.5–6 times higher than the control (Maligeppagol et al., 2013).

8. Conclusions

The update of the literature reinforced the conclusions made in C/NL/09/01 and C/NL/09/02 that the import of flowers of transgenic carnation varieties IFD-26407-2 and IFD-25958-3 will not have any deleterious effects on human health; 1. There is a long history of safe use of non-GM varieties of the parental organism, the carnation. • Carnation extracts have been used as traditional medicines. • Carnation is not used a s a food or food additive • Allergic reactions to carnation are very rare, despite billions of flowers being handled each year. • Carnation is non-poisonous and non-toxic 2. The genes inserted in IFD-26407-2 and IFD-25958-3 are known plant genes, ubiquitous in nature and common in the human diet. • No homology to allergen or toxin proteins was identified through bioinformatic analysis of the nucleotide or amino acid of the sequences flanking the inserted DNA in either IFD- 25958-3 or IFD-26407-2. • No homology to allergen or toxin proteins was identified through bioinformatic analysis of the ORFs generated from the sequences flanking the inserted DNA in either event. • Bioinformatic analysis showed the inserted T-DNA components showed no significant homology to known toxin or allergen genes.. • The gm-hra gene, a modified soybean ALS gene, has mutations analogous to those of SuRB. Transgenic plants containing gm-hra have been shown to be safe in several animal feeding studies. 3. The proteins encoded by the genes inserted in IFD-26407-2 and IFD-25958-3 are known and are common plant proteins, ubiquitous in nature and common in the human diet. • The flavonoid 3'5'- hydroxylase protein is found in all plant foods producing the anthocyanin delphinidin. • The SuRB gene is a mutation of an enzyme found in all plants. • The DFR protein is found in all plant foods containing anthocyanins. • Cytochrome b5 proteins are found in plants, animals, fungi and some bacterial species. • Bioinformatic analysis showed no significant homology of any of the proteins translated from the inserted genes to known toxin or allergen proteins. • The modified proteins retain their original biological function and this function is found in related proteins that have a history of safe use in food. As the exposure level in flowers of transgenic carnation is less than functionally related proteins (in food), then the modified

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protein may be considered to be ‘‘as-safe-as’’ those with a history of safe use (Hammond and Jez, 2011; Hammond et al., 2013). 4. Delphinidin, the accumulation of which is the primary novel trait in IFD-26407-2 and IFD- 25958-3, does not present a potential risk to human health. • Delphinidin is found in numerous ornamental and food plants. • Delphinidin is a component of the human diet, as it is a component of common food plants. • Delphinidin is not a toxic compound and there is no evidence the compound is allergenic. • The scientific literature shows anthocyanins in general, and delphinidin specifically, are beneficial for human health. • Several scientific reports show delphinidin to have anti-cancer, anti-inflammatory effect and other specific health benefits. 5. It is very unlikely IFD-26407-2 and IFD-25958-3 will be used in the manufacture of perfumes or as a food additive. 6. It is very unlikely IFD-26407-2 and IFD-25958-3 will be used as food. If flower petals were used as a garnish there is no reason to believe the inserted genes, the translated proteins from these genes or delphinidin will pose any short or long term hazards to human health.

9. Literature cited

1. Aburjai T and Natsheh FM, 2003. Plants used in cosmetics. Phototherapy Research 17, 987 – 1000. PDF 2. Adams M, Schneider VS., Kluge M, Kessler M and Hamburger M, 2012. Epilepsy in the renaissance: a survey of remedies from 16th and 17th century German herbals. Journal of Ethnopharmacology 143, 1-13. PDF 3. Afaq F, Zama N, Khan N, Side DEN, Safaris S, Abu Said M and Mehta J, 2008. Inhibition of epidermal growth factor receptor signalling pathway by delphinidin, an anthocyanidin in pigmented fruits and vegetables. International Journal of Cancer 123, 1508 – 1515. PDF 4. Anantharaman A, Subramanian B, Chandrasekaran R, Seenivasan R and Siva R, 2014. Colorants and cancer: A review. Industrial Crops and Products 53,167-186. PDF 5. Appenzeller LM, Munley SM, Hoban D, Sykes GP, Malley LA and Delaney B, 2008. Subchronic feeding study of herbicide-tolerant soybean DP-356043-5 in Sprague-Dawley rats. Food and Chemical Toxicology 46, 2201 – 2213. PDF 6. Appenzeller LM, Malley L, Hoban D, Sykes GP and Malley LA, 2009. Subchronic feeding study of grain from herbicide-tolerant maize DP-098140-6 in Sprague-Dawly rats. Food and Chemical Toxicology 47, 2269 – 2280. PDF 7. Azevedo L, de Lima PL. Gomes JC, Strinheta PC, Ribeiro DA and Salvadori DMF, 2007. Differential response related to genotoxicity between eggplant (Solanum melanogena) skin aqueous extract and its main purified anthocyanin (delphinidin) in vivo. Food and Chemical Toxicology 45, 852-858. PDF 8. Barakate A, Shoman SA, Dina N and Alfarouk OR, 2010. Antiviral activity and mode of action of Dianthus caryophyllus L. and Lupinus termes L. seed extracts against in vitro herpes simplex and hepatitis A viruses infection. Journal of Microbiology and Antimicrobials 2, 23-29. PDF 9. Bartholomaeus A, Parrott W, Bondy G and Walker K, 2013. The use of whole food animal studies in the safety assessment of genetically modified crops: limitations and recommendations. Critical Reviews in Toxicology 43, S2, 1-24. PDF 10. Baur X, 2009. Airborne allergens and irritants in the workplace. In allergy and allergic diseases volume 1. Eds Kay AB, Kaplan AP, Bousquet J and Holt PG. Wiley-Blackwell, Oxford, 1017 – 1122. PDF 11. Beecher GR, 2003. Overview of dietary flavonoids: nomenclature, occurrence and intake. Journal of Nutrition 133, 3248S–3254S. PDF 19

Copies of literature reviews

12. Blanco E, Macia MJ and Morales R, 1999. Medicinal and veterinary plants of El Caurel (Galicia, northwest Spain). Journal of Ethnopharmacology 65, 113 – 124. PDF 13. Bolognesi A, 2002. Micronucleus monitoring of a floriculturist population from western Liguria, Italy. Mutagenesis 17, 391-397. PDF 14. Bornsek SM, Ziberna L, Polak T, Vanzo A, Ulrih NP, Abram V, Tramer F and Passamonti S, 2012. Bilberry and blueberry anthocyanins act as powerful intracellular antioxidants in mammalian cells. Food Chemistry 134, 1878-1884. PDF 15. Brinia A, Vovolis V, Tsiougkos N, Petrodimopoulou M and Kompoti E, 2013. Respiratory allergy related to accidental exposure to carnation (Dianthus caryophyllus) in a healthy non atopic patient. Allergy 68, Suppl. 97, p.421. PDF 16. Broadhurst L, 2001. Phytochemicals: the ties that bind. Nutrition Science News July 2001. PDF 17. Brouwer DH, Brouwer R, de Mik G, Maas CL and van Hemmen JJ, 1992. Pesticides in the cultivation of carnations in greenhouses: part 1 –exposure and concomitant health risk. Am Ind Hyg Assoc Journal 53, 575 – 581. PDF 18. Brugliera F, Tao G, Tems U, Kalc G, Mouradova E, Price K, Stevenson K, Nakamura N, Stacey I, Katsumoto Y, Tanaka Y and Mason J, 2013. Violet/blue chrysanthemums— metabolic engineering of the anthocyanin biosynthetic pathway results in novel petal colors. Plant Cell Physiology 54, 1696 – 1710. PDF 19. Buathong R, Saetiew K, Phansiri S, Parinthawong N and Arunyanart S, 2013. Tissue culture and transformation of the antisense DFR gene into lotus (Nelumbo nucifera Gaertn.) through particle bombardment. Scientia Horticulturae 161, 216-222. PDF 20. Bueno JM, Ramos-Escudero F, Saez-Plaza P, Munoz AM, Navas MJ and Asuero AG, 2012a. Analysis and antioxidant capacity of anthocyanin pigments. Part 1: general considerations concerning polyphenols and flavonoids. Critical Reviews in Analytical Chemistry 42, 102 – 125. PDF 21. Bueno JM, Saez-Plaza P, Ramos-Escudero F, Jimenez AM, Fett R and Asuero AG, 2012b. Analysis and antioxidant capacity of anthocyanin pigments. Part 2: chemical structure, color, and intake of anthocyanins. Critical reviews in Analytical Chemistry 42, 126 – 151. PDF 22. Bunea A, Rugina D, Sconta Z, Pop RM, Pintea A, Socaciu C, Tabaran F, Grootaert C, Struijs K and XavCamp J, 2013. Anthocyanin determination in blueberry extracts from various cultivars and their antiproliferative and apoptotic properties in B16-F10 metastatic murine melanoma cells. Phytochemistry 95, 436 – 444. PDF 23. Bushey DF, Bannon GA, Delaney BF, Graser G, Hefford M, Jiang X, Lee TC, Madduri KM, Pariza M, Privalle LS, Ranjan R, Saab-Rincon G, Schafer BW, Thelen JJ, Zhang JX and Harper MS, 2014. Characteristics and safety assessment of intractable proteins in genetically modified crops. Regulatory Toxicology and Pharmacology 69,154-170. PDF 24. Bussmann RW, Malca G, Glenn A, Sharon D, Nilsen B, Parris B, Dubose D, Ruiz D, Saleda J, Martinez, M, Carillo L, Walker K, Kuhlman, A and Townesmith A, 2011. Toxicity of medicinal plants used in traditional medicine in northern Peru. Journal of Ethnopharmacology 137, 121-140. PDF 25. Butelli E, Titta L, Giogio M, Mock H-P, Matros A, Peterk S, Schijlen E, Hall RD, Bovy AG, Luo J and Martin C, 2008. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nature Biotechnology 11, 1301 – 1308. PDF 26. CBI, 2009. Natural ingredients for cosmetics; The EU market for essential oils for cosmetics. CBI product survey, Sep 2009. PDF 27. CBI, 2010. The natural colours, flavours and thickeners market in the EU, Mar 2010. PDF 28. Ceron JJ, Hernandez F, Madrid J and Gutierrez C, 1996. Chemical composition and nutritive value of fresh and ensiled carnation (Dianthus caryophyllus) by-product. Small Ruminant

20

Copies of literature reviews

Research 20, 109 – 112. PDF 29. Céspedes CL, Valdez-Morales M, Avila JG, El-Hafidi M, Alarcón J and Paredes-López O, 2010. Phytochemical profile and the antioxidant activity of Chilean wild black-berry fruits, Aristotelia chilensis (Mol) Stuntz (Elaeocarpaceae). Food Chemistry 119,886-895. PDF 30. Chamcheu JC, Afaq F, Syed DN, Siddiqui IA, Adhami VM, Khan N, Singh S, Boylan BT, Wood GS and Mukhtar H, 2013. Delphinidin, a dietary antioxidant, induces human epidermal keratinocyte differentiation but not apoptosis: studies in submerged and three- dimensional epidermal equivalent models. Experimental Dermatology 22, 342-348. PDF 31. Chen C-Y, Yi L, Jin X, Mi M-T, Zhang T, Ling W-H and Yu B. 2010, Delphinidin attenuates stress injury induced by oxidized low-density lipoprotein in human umbilical vein endothelial cells. Chemico-Biological interactions 183, 105 – 112. PDF 32. Chiang A-N, Wu H-L, Yeh HI, Chu C-S, Lin H-C and Lee W-C, 2006. Antioxidant effects of black rice extract through the induction of superoxide dismutase and catalase activities. Lipids 41, 797–803. PDF 33. Chukwudbe A, Privalle L, Reed A, Wandelt C, Contri D, Dammann M, Groeters S, Kaspers U, Strauss V and van Ravenzwaay B, 2012. Health and nutritional status of Wistar rats following subchronic exposure to CV127 soybeans. Food and Chemical Toxicology 50, 956 – 971. PDF 34. Chun OK, Lee SG, Wang Y, Vance T and Song WO, 2012. Estimated flavonoid intake of the elderly in the United States and around the world. Journal of Nutrition in Gerontology and Geriatrics 31, 190 – 205. PDF 35. Cistero-Bahima A, Enrique E, Alonso R, Del Marsam Miguel, M and Bartolom B, 2000. Simultaneous occupational allergy to carnation and its parasite in a glasshouse worker. Journal of Allergy and Clinical Immunology 106, p.780. PDF 36. Corcoran MP, 2012. Flavonoid basics: chemistry, sources, mechanisms of action, and safety. Journal of nutrition in Gerontology and Geriatrics 31, 176 – 189. PDF 37. CPPIS (Canadian Poisonous Plants Information System), Online. Database of poisonous plants available at http://www.cbif.gc.ca/eng/species-bank/canadian-poisonous-plants-information- system/?id=1370403265036 38. Crespy V, Morand C, Besson C, Cotelle N, Herve V, Demigne C and Remesy C, 2003. The splanchnic metabolism of flavonoids highly differed according to the nature of the compound. American Journal of Physiology – Gastrointestinal Liver Physiology 284, G980–G988. PDF 39. Cui C, Zhang S, You L, Ren J, Luo W, Chen W and Zhao M, 2013. Antioxidant capacity of anthocyanins from Rhodomyrtus tomentosa (Ait.) and identification of the major anthocyanins. Food Chemistry 139, 1-8. PDF 40. Cvorovic J, Tramer F, Granzotto M, Candussio L, Decorti G and Passamonti S, 2010. Oxidative stress-based cytotoxicity of delphinidin and cyanidin in colon cancer cells. Archives of Biochemistry and Biophysics 501, 151 – 157. PDF 41. Dayoub O, Andriantsitohaina R and Clere N, 2013. Pleiotropic beneficial effects of epigallocatechin gallate, quercetin and delphinidin on cardiovascular diseases associated with endothelial dysfunction. Cardiovascular Hematological Agents Medicinal Chemistry 11, 249-264. PDF 42. Delaney B, Appenzeller LM, Munley SM, Hoban D, Sykes GP, Malley LA and Sanders C, 2008. Subchronic feeding study of high oleic acid soybeans (Event DP-305423-1) in Sprague-Dawley rats. Food and Chemical Toxicology 46, 3808 – 3817. PDF 43. Ding C, Zhang W, Li J, Lei J and Yu J. 2013. Cytotoxic constituents of ethyl acetate fraction from Dianthus superbus. Natural Product Research 27, 1691-1694. PDF 44. Dixon RA, Liu C and Jun JH, 2013. Metabolic engineering of anthocyanins and condensed tannins in plants. Current Opinions in Biotechnology 24,329-335. PDF

21

Copies of literature reviews

45. EC, 1997. Opinion on colours in foods for special medical purposes for young children. In: European Commission. Reports of the Scientific Committee for food (forty-first series), Office for Official Publications of the European Communities, Luxembourg 1-8. PDF 46. Eriksson NE, Moller C, Werner S, Magnusson J, Bengtsson U and Zolubas M, 2004. Self- reported food hypersensitivity in Sweden, Denmark, Estonia, Lithuania and Russia. Journal of Investigational Allergology and Clinical Immunology 14, 70 -79. PDF 47. EU (European Union), Online, a. The catalogue of novel food products in the EU available at http://ec.europa.eu/food/food/biotechnology/novelfood/novel_food_catalogue_en.htm 48. EU (European Union), Online, b. Novel foods and novel food ingredients that may be placed on the market in the EU pursuant to Regulation (EC) No. 258/97 available at http://ec.europa.eu/food/food/biotechnology/novelfood/authorisations_en.htm 49. EU (European Union), Online, c. The database of food additives authorized in the EU available at https://webgate.ec.europa.eu/sanco_foods/main/ 50. Ezuruike UF and Prieto JM, 2014. The use of plants in the traditional management of diabetes in Nigeria: Pharmacological and toxicological considerations. Journal Ethnopharmacology http://dx.doi.org/10.1016/j.jep.2014.05.055. PDF 51. FAARP (Food Allergy and Resource Program), Online. Available at www.allergenonline.com 52. Felgines C, Texier O, Morand C, Manach C, Scalbert A, Great F and Reme C, 2000. Bioavailability of the flavanone naringenin and its glycosides in rats American Journal of Physiology – Gastrointestinal Liver Physiology 279, G1148–G1154. PDF 53. Feng R, Wang SY, Shi YH, Fan J and Yin XM, 2010. Delphinidin induces necrosis in hepatocellular carcinoma cells in the presence of 3-methyladenine, an autophagy inhibitor. Journal of Agricultural and Food Chemistry 58, 3957-3964. PDF 54. Fernandes I, Nave F, Goncalves R, de Freitas V and Mateus N, 2012. On the bioavailability of flavanols and anthocyanins: flavanol-anthocyanin dimers. Food Chemistry 135, 812-818. PDF 55. Fernandes I, Marques F, de Freitas V and Mateus N, 2013. Antioxidant and antiproliferative properties of methylated metabolites of anthocyanins. Food Chemistry 141, 2923-2933. PDF 56. Fernandes I, Faria A, Calhau C, de Freitas V and Mateus N, 2014. Bioavailability of anthocyanins and derivatives. Journal of Functional Foods 7, 54-66. PDF 57. Fukui Y, Tanaka Y, Kusumi T, Iwashita T and Nomoto K, 2003. A rationale for the shift in colour towards blue in transgenic carnation flowers expressing the flavonoid 3’, 5’- hydroxylase gene. Phytochemistry 63, 15–23. PDF 58. Galeotti F, Barile E, Curir P, Dolci M and Lanzotti V, 2008. Flavonoids from carnation (Dianthus caryophyllus) and their antifungal activity. Phytochemistry Letters 1, 44-48. PDF 59. Garcia-Estevez I, Jacquet R, Alcalde-Eon C, Rivas-Gonzalo JC, Escribano-Bailon MT and Quideau S, 2013. Hemisynthesis and structural and chromatic characterization of delphinidin 3-O-glucoside-vescalagin hybrid pigments. Journal Agricultural and Food Chemistry 61, 11560-11568. PDF 60. Gevrenova R, Joubert O, Mandova T, Zaiou M, Chapleur Y and Henry M, 2014. Cytotoxic effects of four caryophyllaceae species extracts on macrophage cell lines. Pharmaceutical Biology 52, 919-925. PDF 61. Gonzali S, Mazzucato A and Perata P, 2009. Purple as a tomato: towards high anthocyanin tomatoes. Trends in Plant Science 14, 237 – 241. PDF 62. Gou J, Zou Y and Ahn J, 2011. Enhancement of antioxidant and antimicrobial activities of Dianthus superbus, Polygonum aviculare, Sophora flavescens, and Lygodium japonicum by pressure-assisted water extraction. Food Science and Biotechnology 20, 283-287. PDF 63. Guo Y and Qiu L, 2013. Allele specific marker development and selection efficiencies for both flavonoid 3’-hydroxylase and flavonoid 3’-5’-hydroxylase genes in soybean subgenus soja. Theoretical and Applied Genetics 126, 1445 – 1455. PDF

22

Copies of literature reviews

64. Guzmán R, Santiago C and Sánchez M, 2009. A density functional study of antioxidant properties on anthocyanidins. Journal of Molecular Structure 935,110-114. PDF 65. Hafeez BB, Siddiqui IA, Asim M, Malik A, Afaq F, Adhami, VM, Saleem M, Din M and Mukhtar H, 2008. A dietary anthocyanidin delphinidin induces apoptosis of human prostate cancer PC3 cells in vitro and in vivo: involvement of nuclear factor-ĸB signaling. Cancer Research 68, 8564 – 8572. PDF 66. Hammond BG and Jez JM, 2011. Impact of food processing on the safety assessment for proteins introduced into biotechnology-derived soybean and corn crops. Food and Chemical Toxicology 49,711-721. PDF 67. Hammond B, Kough J, Herouet-Guicheney C and Jez JM, 2013. Toxicological evaluation of proteins introduced into food crops. Critical Reviews in Toxicology 43 Suppl 2, 25-42. PDF 68. Hanif S, Shamim U, Ullah MF, Azmi AS, Bhat SH and Hadi SM, 2008. The anthocyanidin delphinidin mobilizes endogenous copper ions from human lymphocytes leading to oxidative degradation of cellular DNA. Toxicology 249, 19-25. PDF 69. Harvey JH, Gilliflower and carnation. 1978. Garden History 6, 46 – 57. PDF 70. Haseeb A, Chen D and Haqqi TM, 2012. Delphinidin blocks IL-1β-induced activation of NF-κB by modulating the IKKβ gene expression and the activation of NIK in human chondrocytes. Osteoarthritis and Cartilage 20, p.S146. PDF 71. Haseeb A, Chen D and Haqqi TM, 2013. Delphinidin inhibits IL-1beta-induced activation of NF-kappaB by modulating the phosphorylation of IRAK-1(Ser376) in human articular chondrocytes. Rheumatology 52, 998-1008. PDF 72. Heinonen M, 2007. Review; antioxidant activity and antimicrobial effect of berry phenolics – a Finnish perspective. Molecular Nutrition and Food Research 51, 684–691. PDF 73. Hou D-X, Yanagita T, Uto T, Masuzaki S and Fujii M, 2005. Anthocyanidins inhibit cyclooxygenase-2 expression in LPS-evoked macrophages: structure-activity relationship and molecular mechanisms involved. Biochemical Pharmacology 70, 417–425. PDF 74. Huang W, Sun W and Wang Y, 2012. Isolation and molecular characterisation of flavonoid 3'-hydroxylase and flavonoid 3', 5'-hydroxylase genes from a traditional Chinese medicinal plant, Epimedium sagittatum. Gene 497,125-130. PDF 75. Hwang MK, Kang NJ, Heo YS, Lee KW and Lee HJ, 2009. Fyn kinase is a direct molecular target of delphinidin for the inhibition of cyclooxygenase-2 expression induced by tumor necrosis factor-alpha. Biochemical Pharmacology 77, 1213-1222. PDF 76. Inomata Y, Terahara N, Kitajima J, Kokubugata G and Iwashina T, 2013. Flavones and anthocyanins from the leaves and flowers of Japanese Ajuga species (Lamiaceae). Biochemical Systematics and Ecology 51,123-129. PDF 77. Ishiguro K, Taniguchi M and Tanaka Y, 2012. Functional analysis of Antirrhinum kelloggii flavonoid 3'-hydroxylase and flavonoid 3', 5'-hydroxylase genes; critical role in flower color and evolution in the genus Antirrhinum. Journal of Plant Research 125,451-456. PDF 78. Jara E, Hidalgo MA, Hancke JL, Hidalgo AI, Brauchi S, Nunez L, Villalobos C and Burgos RA, 2014. Delphinidin activates NFAT and induces IL-2 production through SOCE in T cells. Cell Biochemistry and Biophysics 68,497-509. PDF 79. Jin X, Yi L, Chen M-L, Chen C-Y, Chang H, Zhang T, Wang L, Zhu J-D, Zhang Q-Y and Mi M-T, 2013. Delphinidin-3-glucoside protects against oxidized low-density lipoprotein- induced mitochondrial dysfunction in vascular endothelial cells via the sodium-dependent glucose transporter SGLT1. . PLoS ONE 5(8), e12441 8:e68617. PDF 80. Johnson MH, de Mejia EG, Fan J, Lila MA and Yousef GG, 2013. Anthocyanins and proanthocyanidins from blueberry-blackberry fermented beverages inhibit markers of inflammation in macrophages and carbohydrate-utilizing enzymes in vitro. Molecular Nutrition and Food Research 57, 1182-1197. PDF 81. Kalt W, McDonald JE, Ricker RD and Lu X, 1999. Anthocyanin content and profile within and among blueberry species. Canadian Journal of Plant Science 79, 617-623. PDF

23

Copies of literature reviews

82. Kamonpatana K, Giusti MM, Chitchumroonchokchai C, Moreno Cruz M, Ried KM, Kumar P and Failla ML, 2012. Susceptibility of anthocyanins to ex vivo degradation in human saliva. Food Chemistry 135, 738–747. PDF 83. Kausar H, Jeyabalan J, Aqil F, Chabba D, Sidana J, Singh IP and Gupta RC, 2012. Berry anthocyanidins synergistically suppress growth and invasive potential of human non-small- cell lung cancer cells. Cancer Letters 325, 54-62. PDF 84. Khan NQ, Lees DM, Douthwaite JA, Carrier MJ and Corder R, 2002. Comparison of red wine extract and polyphenol constituents on endothelin-1 synthesis by cultured endothelial cells. Clinical Science 103, suppl. 48, 72S–75S. PDF 85. Kim HK, Kim JN, Han SN, Nam JH, Na HN, and Ha TJ, 2012a. Black soybean anthocyanins inhibit adipocyte differentiation in 3T3-L1 cells. Nutrition Research 32,770- 777. PDF 86. Kim SM, Chung MJ, Ha TJ, Choi HN, Jang SJ, Kim SO, Chun MH, Do SI, Choo YK and Park YI, 2012b. Neuroprotective effects of black soybean anthocyanins via inactivation of ASK1-JNK/p38 pathways and mobilization of cellular sialic acids. Life Sciences 90,874- 882. PDF 87. Kimbaris AC, Koliopoulos G, Michaelakis A, and Konstantopoulou MA, 2012. Bioactivity of Dianthus caryophyllus, Lepidium sativum, Pimpinella anisum, and Illicium verum essential oils and their major components against the west Nile vector Culex pipiens. Parasitology research 111, 2403-2410. PDF 88. Koh K, Youn JE and Kim HS, 2014. Identification of anthocyanins in black soybean (Glycine max (L.) Merr.) varieties. Journal of Food Science and Technology 51, 377-381. PDF 89. Kruger MJ, Davies N, Myburgh KH and Lecour S, 2014. Proanthocyanidins, anthocyanins and cardiovascular diseases. Food Research International 59, 41-52. PDF 90. Lamminpaa A, Estlander T, Jolanki R and Kanerva L, 1996. Occupational allergic contact dermatitis caused by decorative plants. Contact Dermatitis 34, 330 -335. PDF 91. Lamula SQN and Ashafa AOT, 2014. Antimicrobial and cytotoxic potential of Dianthus basuticus used in Basotho traditional practice. Bangladesh Journal of Pharmacology 9, 105- 111. PDF 92. Lamy S, Blanchette M, Michaud-Levesque J, Lafeur R, Durocher Y, Moghrabi A, Barrette S, Gingras D and Beliveau R, 2006. Delphinidin, a dietary anthocyanidin, inhibits vascular endothelial growth factor receptor-2 phosphorylation. Carcinogenesis 27, 989–996. PDF 93. Lamy S, Lafleur R, Bedard V, Moghrabi A, Barrette S, Gingra D and Beliveaul R, 2007. Anthocyanidins inhibit migration of glioblastoma cells: structure-activity relationship and involvement of the plasminolytic system. Journal of Cellular Biochemistry 100, 100–111. PDF 94. Lazze MC, Pizzala R, Perucca P, Cazzalini O, Savio M, Forti L, Vannini V and Bianchi L, 2006. Anthocyanidins decrease endothelin-1 production and increase endothelial nitric oxide synthase in human endothelial cells Molecular Nutrition and Food Research 50, 44–51. PDF 95. León-González AJ, Truchado P, Tomás-Barberán FA, López-Lázaro M, Barradas MCD and Martín-Cordero C, 2013. Phenolic acids, flavonols and anthocyanins in Corema album (L.) D. Don berries. Journal of Food Composition Analysis 29, 58-63. PDF 96. Li H, Deng Z, Zhu H, Hu C, Liu R, Young JC and Tsao R, 2012. Highly pigmented vegetables: anthocyanin compositions and their role in antioxidant activities. Food Research International 46,250-259. PDF 97. Lila S, 2004. Anthocyanins and human health: an in vitro investigative approach. Journal of Biomedical Biotechnology 5, 306–313. PDF 98. Lim TG, Jung SK, Kim JE, Kim Y, Lee HJ, Jang TS and Lee KW, 2013. NADPH oxidase is a novel target of delphinidin for the inhibition of UVB-induced MMP-1 expression in human dermal fibroblasts. Experimental Dermatology 22,428-430. PDF

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Copies of literature reviews

99. Lim TK, 2014. Dianthus caryophyllus. In: Edible medicinal and non-medicinal plants Ed. Lim TK. Springer, Dordrecht, Netherlands, 684-693. PDF 100. Lin YC, Tsai PF and Wu JS, 2014. Protective effect of anthocyanidins against sodium dithionite-induced hypoxia injury in C6 glial cells. Journal of Agricultural and Food Chemistry. 62, 5603−5608. PDF 101. Liu N, Sun G, Xu Y, Luo Z, Lin Q, Li X, Zhang J and Wang L, 2013. Anthocyanins of the genus of Hosta and their impacts on tepal colors. Scientia Horticulturae 150,172-180. PDF 102. Liu S, Ju J and Xia G, 2014. Identification of the flavonoid 3'-hydroxylase and flavonoid 3', 5'-hydroxylase genes from Antarctic moss and their regulation during abiotic stress. Gene 543,145-152. PDF 103. López-Expósito1 I, Castillo A, Yang N, Liang B and Li X-M, 2011. Chinese herbal extracts of Rubia cordifolia and Dianthus superbus suppress IgE production and prevent peanut-induced anaphylaxis. Chinese Medicine 6, p.35. PDF 104. Lou Q, Liu Y, Qi Y, Jiao S, Tian F, Jiang L and Wang Y. 2014, Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth. Journal of Experimental Botany 65, 3157-3164. PDF 105. Lubbe A and Verpoorte R 2011, Cultivation of medicinal and aromatic plants for specialty industrial materials. Industrial Crops and Products 34, 785 – 801. PDF 106. Lucas, CD, Hallagan, JB and Taylor S, 2001. The role of natural color additives in food allergy. Advances in Food and Nutrition Research 43, 195-216. PDF 107. Maligeppagol M, Chandra1 GS, Prakash M, Navale H, Deepa PR, Rajeev R, Asokan K, Babu PCS, Babu B, Rao VK and Kumar NKK, 2013. Anthocyanin enrichment of tomato (Solanum lycopersicum L.) fruit by metabolic engineering. Current Science 105, 72 - 80. PDF 108. Manach C, Williamson G, Morand C, Scalbert A and Remesy C, 2005. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. American Journal of Clinical Nutrition 81, suppl, 230S–42S. PDF 109. Martin C, Zhang Y, Tonelli, C and Petroni K, 2013. Plants, diet and health. Annual Review of Plant Biology 64, 19 – 46. PDF 110. Martin S, Favot L, Matz R, Lugnier C. and Andriantsitohaina R, 2003. Delphinidin inhibits endothelial cell proliferation and cell cycle progression through a transient activation of ERK-1/-2. Biochemical Pharmacology 65, 669-75. PDF 111. Martineti V, Tognarini I, Azzari C, Carbonell Sala S, Clematis F, Dolci M, Lanzotti V, Tonelli F, Brandi ML and Curir P, 2010. Inhibition of in vitro growth and arrest in the G0/G1 phase of HCT8 line human colon cancer cells by kaempferide triglycoside from Dianthus caryophyllus. Phytotherapy Research 24, 1302-1308. PDF 112. Mathesius CA, Barnett JF, Cressman RF, Ding J, Carpenter C, Ladics GS, Schmist J, Layton RJ, Zhang JXQ, Appenzeller LM, Carlson G, Ballou S and Delaney B, 2009. Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans. Regulatory Toxicology and Pharmacology 55, 309 – 320. PDF 113. Matheu V, Baeza ML, Zubeldia JM and Barrios Y, 2004. Allergy to lingonberry: A case report. Clinical and Molecular Allergy 2, p.2. PDF 114. Matsubara K Kaneyuki T Miyake T and Mori M, 2005. Antiangiogenic activity of nasunin, an antioxidant anthocyanin, in eggplant peels. Journal of Agricultural and Food Chemistry 53, 6272-6275. PDF 115. Mazzucato A, Willems D, Bernini R, Picarella ME, Santangelo E, Ruiu F, Tilesi F and Soressi GP, 2013. Novel phenotypes related to the breeding of purple-fruited tomatoes and effect of peel extracts on human cancer cell proliferation. Plant Physiology and Biochemistry 72,125-133. PDF

25

Copies of literature reviews

116. McNaughton J, Roberts M, Rice D, Smith B, Hinds M, Delaney B, Iiams C and Sauber T, 2011a. Nutritional equivalency evaluation of transgenic maize grain from event DP-098140-6 and transgenic soybeans containing event DP-356043-5: laying hen performance and egg quality measures. Poultry Science 90, 377 – 389. PDF 117. McNaughton J, Roberts M, Rice D, Smith B, Hinds M, Delaney B, Iiams C and Sauber T, 2011b. Comparison of broiler performance and carcass yields when fed transgenic maize grain containing event DP-098140-6 and processed fractions from transgenic soybeans containing event DP-356043-5. Poultry Science 90, 1701 – 1711. PDF 118. Meija L, Jacobs CM, Utterback PL, Parsons CM, Rice D, Sanders C, Smith,B, Iiams C and Sauber T, 2010. Evaluation of the nutritional equivalency of soybean meal with the genetically modified trait DP-305423-1 when fed to laying hens. Poultry Science 89, 2634 – 2639. PDF 119. Miladinovic B, Kostic M, Savikin K, Dordevic B, Mihajilov-Krstev T, Zivanovic S and Kitic D, 2014. Chemical profile and antioxidative and antimicrobial activity of juices and extracts of 4 black currants varieties (Ribes nigrum L.). Journal of Food Science 79, C301-309. PDF 120. Miyagawa N, Nishizaki Y, Miyahara T, Okamoto M, Hirose Y, Ozeki Y and Sasaki N, 2014. Sequence variations in the flavonoid 3’5’-hydroxylase gene associated with reddish flower phenotypes in three delphinium varieties. Plant Biotechnology 31, 83-87. PDF 121. Mlcek J and Rop O, 2011. Fresh edible flowers of ornamental plants – a new source of nutraceutical foods. Trends in Food Science and Technology 22, 561 – 569. PDF 122. More DR, Napoli DC and Hagan LL, 2003. Herbal supplements and skin testing: the lack of effect of commonly used herbal supplements on histamine skin prick testing Allergy 58, 492–494. PDF 123. Moreau C, Ambrose MJ, Turner L, Hill L, Ellis TH and Hofer JM, 2012. The B gene of pea encodes a defective flavonoid 3', 5'-hydroxylase, and confers pink flower color. Plant Physiology 159,759-768. PDF 124. Mori S, Otani M, Kobayashi H and Nakano M, 2014. Isolation and characterization of the dihydroflavonol 4-reductase gene in the monocotyledonous ornamental Agapanthus praecox ssp. orientalis (Leighton) Leighton. Scientia Horticulturae 166, 24-30. PDF 125. Moriwaki S, Suzuki K, Muramatsu M, Nomura A, Inoue F, Into T, Yoshiko Y and Nida S, 2014. Delphinidin, one of the major anthocyanidins, prevents bone loss through the inhibition of excessive osteoclastogenesis in osteoporosis model mice. PLoS ONE 5 8, e12441 9:e97177. PDF 126. Murapa P, Dai J, Chung M, Mumper RJ and D’Orazio J, 2012. Anthocyanin-rich fractions of blackberry extracts reduce UV-induced free radicals and oxidative damage in kerationcytes. Phytotherapy Research 26, 106 – 112. PDF 127. Muthanna JM and Al-Bayati A, 2009. Isolation and identification of antibacterial compounds from Thymus kotsyhanus aerial parts and Dianthus caryophyllus flower buds. Phytomedicine 16, 632 – 637. PDF 128. Nakamura N, Fukuchi-Mizutani M, Katsumoto Y, Togami J, Senior M, Matsuda Y, Furuichi K, Yoshimoto M, Matsunaga A, Ishiguro K, Aida M, Tasaka M, Fukui H, Tsuda S, Chandler S and Tanaka Y, 2011 Environmental risk assessment and field performance of rose genetically modified for delphinidin production. Plant Biotechnology 28, 251–261. PDF 129. Navarro AM, Sanchez, MC, Orta JC, Delgado J and Conde J, 2001. Tetranychids and occupational allergy. Alergologia e Immunologia Clinica 16, 5-10. PDF 130. NCSU (North Carolina State University), Online. Carnation entry in database of poisonous plants of North Carolina available at http://plants.ces.ncsu.edu/plants/edible/dianthus-spp/

26

Copies of literature reviews

131. Nichenametla SN, Taruscio TG, Barney DL and Exon JH, 2006. A review of the effects and mechanisms of polyphenolics in cancer. Critical Reviews in Food Science and Nutrition 46, 161–183. PDF 132. Nitteranon V, Kittiwongwattana C, Vuttipongchaikij S, Sakulkoo J, Srijakkoat M, Chokratin P, Harinasut P, Suputtitada S and Apisitwanich S, 2014. Evaluations of the mutagenicity of a pigment extract from bulb culture of Hippeastrum reticulatum. Food and Chemical Toxicology 69, 237-243. PDF 133. Noda N, Aida R, Kishimoto S, Ishiguro K, Fukuchi-Mizutani M, Tanaka Y and Ohmiya A, 2013. Genetic engineering of novel bluer-colored chrysanthemums produced by accumulation of delphinidin-based anthocyanins. Plant and Cell Physiology 54, 1684-1695 PDF 134. Noda Y, Kaneyuki T, Mori A and Packer L, 2002. Antioxidant activities of pomegranate fruit extract and its anthocyanidins; delphinidin, cyanidin and pelargonidin. Journal of Agricultural and Food Chemistry 50, 166–171. PDF 135. Norberto S, Silva S, Meireles M, Faria A, Pintado M, and Calhau C, 2013. Blueberry anthocyanins in health promotion: a metabolic overview. Journal of Functional Foods 5, 1518-1528. PDF 136. Ogawa K, Tsuruma K, Tanaka J, Kakino M, Kobayashi S, Shimazawa M and Hara H, 2013. The protective effects of bilberry and lingonberry extracts against UV light- induced retinal photoreceptor cell damage in vitro. Journal of Agricultural and Food Chemistry 61, 10345-10353. PDF 137. Ogawa K, Kuse Y, Tsuruma K, Kobayashi S, Shimazawa M and Hara H, 2014. Protective effects of bilberry and lingonberry extracts against blue light-emitting diode light- induced retinal photoreceptor cell damage in vitro. BMC Complementary and Alternative Medicine 14, p.120. PDF 138. OGTR, 2006. The biology and ecology of Dianthus caryophyllus L. (carnation). Office of the gene technology Regulator, Australia. PDF 139. Ohmiya A, Tanase K, Hirashima M, Yamamizo C, Yagi M, and Debener T, 2013. Analysis of carotenogenic gene expression in petals and leaves of carnation (Dianthus caryophyllus L.). Plant Breeding DOI:10.1111/pbr.12061. PDF 140. Ojeda D, Jimenez-Ferrer E, Zamilpa A, Herrera-Arellano A, Tortoriello J and Alvarez L.2010, Inhibition of angiotension convertin enzyme (ACE) activity by the anthocyanins delphinidin- and cyanidin-3-O-sambubiosides from Hibiscus sabdariffa. Journal of Ethnopharmacology 127, 7 – 10. PDF 141. Ono E, Ruike M, Iwashita T, Nomoto K and Fukui Y, 2010. Co-pigmentation and flavonoid glycosyltransferases in blue Veronica persica flowers. Phytochemistry 71,726- 735. PDF 142. Orta JC, Navarro AM, Bartolome B, Delgado J, Martinez J, Sanchez MC, Matinez A, Valverdu A, Conde J and Palacios R, 1998. Comparative allergenic study of Tetranychus urticae from different sources. Journal of Investigational Allergology and Clinical Immunology 8, 149 – 154. PDF 143. Osorio C, Hurtado N, Dawid C, Hofmann T, Heredia-Mira FJ and Morales AL, 2012. Chemical characterisation of anthocyanins in tamarillo (Solanum betaceum Cav.) and Andes berry (Rubus glaucus Benth.) fruits. Food Chemistry 132, 1915-1921. PDF 144. Ozbay T and Nahta R, 2011. Delphinidin Inhibits HER2 and Erk1/2 Signaling and suppresses growth of HER2-over expressing and triple negative breast cancer cell lines. Breast Cancer 5,143-154. PDF 145. Patel K, Jain A and Patel DK, 2013. Medicinal significance, pharmacological activities, and analytical aspects of anthocyanidins ‘delphinidin’: A concise report. Journal of Acute Disease 2,169-178. PDF 146. Perez-Jimenez J, Fezeu L, Touvier M, Arnault N, Manach C, Hercberg S, Galan P

27

Copies of literature reviews

and Scalbert A, 2011. Dietary intake of 337 polyphenols in French adults. The American Journal of Clinical Nutrition 93, 1220 – 1228. PDF and suppl. 147. Pieroni A, Quave CL, Villanelli ML, Mangino P, Sabbatini G, Santini L, Boccetti T, Profili M, Ciccioli T, Rampa LG, Antonini G, Girolamini C, Cecchi M and Tomasi M, 2004. Ethnopharmacognostic survey on the natural ingredients used in folk cosmetics, cosmeceuticals and remedies for healing skin diseases in the inland Marches, central-eastern Italy. Journal of Ethnopharmacology 91, 331–344, 2004. PDF 148. Prior RL and Wu X, 2006. Anthocyanins: Structural characteristics that result in unique metabolic patterns and biological activities. Free Radical Research 40, 1014–1028. PDF 149. Puckhaber LS, Stopanovic RD and Bost GA, 2002. Analyses for flavonoid aglycones in fresh and preserved Hibiscus flowers. In: trends in new crops and new uses. Eds Janick J and Whipkey A. ASHS press, Alexandria, USA, 556–563. PDF 150. Qi, Y, Lou Q, Quan Y, Liu Y and Wang Y, 2013. Flower-specific expression of the Phalaenopsis flavonoid 3’, 5’-hydoxylase modifies flower color pigmentation in petunia and Lilium. Plant Cell, Tissue and Organ Culture 115, 263-273. PDF 151. Quinones M, Miguel M and Aleixandre A, 2013. Beneficial effects of polyphenols on cardiovascular disease. Pharmacological Research 68,125-131. PDF 152. Rop O, Mlcek J, Jurikova T, Neugebauerova J and Vabkova J, 2012. Edible flowers – a new promising source of mineral elements in human nutrition. Molecules 17, 6662 – 6683. PDF 153. Ruiz A, Hermosín-Gutiérrez I, Vergara C, von Baer D, Zapata M, Hitschfeld A, Obando L and Mardones C, 2013. Anthocyanin profiles in south Patagonian wild berries by HPLC-DAD-ESI-MS/MS. Food Research International 51,706-713. PDF 154. Saito N, Tatsuzawa F, Toki K, Shinoda K, Shigihara A and Honda T, 2011. The blue anthocyanin pigments from the blue flowers of Heliophila coronopifolia L. (Brassicaceae). Phytochemistry 72, 2219-2229. PDF 155. Sánchez-Fernández C, González-Gutiérrez ML, Esteban-López MI, Martínez A and Lombardero M. 2004, Occupational asthma caused by carnation (Dianthus caryophyllus) with simultaneous IgE-mediated sensitization to Tetranychus urticae. Allergy 59, 114 – 115. PDF 156. Sanchez-Guerrero IM, Escudero AI, Bartolom B and Palacios R, 1999. Occupational allergy to carnation. Journal of Allergy and Clinical Immunology 104,181-185. PDF 157. Sancho RAS and Pastore GM, 2012. Evaluation of the effects of anthocyanins in type 2 diabetes. Food Research International 46,378-386. PDF 158. Sasaki N and Nakayama T, 2014. Achievements and perspectives in biochemistry concerning anthocyanin modification for blue flower coloration. Plant and Cell Physiology, pcu097. PDF 159. Sasaki N, Matsuba Y, Abe Y, Okamura M, Momose M, Umemoto N, Nakayama M, Itoh Y and Ozeki Y, 2013. Recent advances in understanding the anthocyanin modification steps in carnation flowers. Scientia Horticulturae 163, 37-45. PDF 160. Scarabelli TM, Mariotto S, Abdel-Azeim S, Shoji K, Darra E, Stephanou A, Chen- Scarabelli C, Marechal JD, Knight R, Ciampa A, Saravolatz L, de Prati AC, Yuan Z, Cavalieri E, Menegazzi M, Latchman D, Pizza C, Perahia D and Suzuki H, 2009. Targeting STAT1 by myricetin and delphinidin provides efficient protection of the heart from ischemia/reperfusion-induced injury. FEBS Letters 583,531-541. PDF 161. Seong AR, Yoo JY, Choi K, Lee MH, Lee YH, Lee J, Jun W, Kim S and Yoon HG, 2011. Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-kappaB acetylation in fibroblast-like synoviocyte MH7A cells. Biochemical and Biophysical Research Communications 410,581-586. PDF 162. Shan Q, Zheng Y, Lu J, Zhang Z, Wu D, Fan S, Hu B, Cai X, Cai H, Liu P and Liu

28

Copies of literature reviews

F, 2014. Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice. Food and Chemical Toxicology 69,339-346. PDF 163. Shin J, Choi C, Shim J, Ryu M, Kwon K, Park Seo H, Lee S, Lim D, Cho N and Cho S, 2013. In vitro apoptotic effects of methanol extracts of Dianthus chinensis and Acalypha australis L. targeting specificity protein 1 in human oral cancer cells. Head and Neck 35, 992 - 998. PDF 164. Skemiene K, Rakauskaite G, Trumbeckaite S, Liobikas J, Brown GC and Borutaite V, 2013. Anthocyanins block ischemia-induced apoptosis in the perfused heart and support mitochondrial respiration potentially by reducing cytosolic cytochrome c. International Journal of Biochemistry and Cell Biology 45, 23-29. PDF 165. Son JE, Lee E, Jung SK, Kim JE, Oak MH, Lee KW and Lee HJ, 2014. Anthocyanidins, novel FAK inhibitors, attenuate PDGF-BB-induced aortic smooth muscle cell migration and neointima formation. Cardiovascular Research 101,503-512. PDF 166. Srovnalova A, Svecarova M, Kopecna Zapletalova M, Anzenbacher P, Bachleda P, Anzenbacherova E and Dvorak Z, 2014. Effects of anthocyanidins and anthocyanins on the expression and catalytic activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in primary human hepatocytes and human liver microsomes. Journal of Agricultural and Food Chemistry 62, 789−797. PDF 167. Stefanaki EC and Pitsios C, 2008. Occupational dermatitis because of carnation. Contact Dermatitis 58, 119-120. PDF 168. Sterling M, 2001. Anthocyanins. Nutrition Science News, December 2001. PDF 169. Sun Y, Huang H, Meng L, Hu K and Dai SL, 2013. Isolation and functional analysis of a homolog of flavonoid 3', 5'-hydroxylase gene from Pericallis X hybrida. Physiologia Plantarum 149,151-159. PDF 170. Takasawa R, Saeki K, Tao A, Yoshimori A, Uchiro H, Fujiwara M and Tanuma S, 2010. Delphinidin, a dietary anthocyanidin in berry fruits, inhibits human glyoxalase I. Bioorganic and Medicinal Chemistry 18, 7029-7033. PDF 171. Tanaka J, Kadekaru T, Ogawa K, Hitoe S, Shimoda H and Hara H, 2013. Maqui berry (Aristotelia chilensis) and the constituent delphinidin glycoside inhibit photoreceptor cell death induced by visible light. Food Chemistry 139,129-137. PDF 172. Teller N, Thiele W, Boettler U, Sleeman J and Marko D, 2009. Delphinidin inhibits a broad spectrum of receptor tyrosine kinases of the ErbB and VEGFR family. Molecular Nutrition and Food Research 53, 1075-1083. PDF 173. Thiele W, Rothley M, Teller N, Jung N, Bulat B, Plaumann D, Vanderheiden S, Schmaus A, Cremers N, Goppert B, Dimmler A, Eschbach V, Quagliata L, Thaler S, Marko D, Brase S and Sleeman JP, 2013. Delphinidin is a novel inhibitor of lymphangiogenesis but promotes mammary tumor growth and metastasis formation in syngeneic experimental rats. Carcinogenesis 34, 2804-2813. PDF 174. Trojan V, Musilová M, Vyhnánek T, Klejdus B, Hanáček P and Havel L, 2014. Chalcone synthase expression and pigments deposition in wheat with purple and blue colored caryopsis. Journal of Cereal Science 59, 48-55. PDF 175. Tsuda T, 2012. Dietary anthocyanin-rich plants: biochemical basis and recent progress in heath benefit studies. Molecular Nutrition and Food Research 56, 159 – 170. PDF 176. UA (Univ. of Arizona), Online. College of pharmacy database of poisonous plants. Available at http://www.pharmacy.arizona.edu/centers/arizona-poison-drug-information- center/plantsgood 177. Ugulu I, 2012. Fidelity level and knowledge of medicinal plants used to make therapeutic Turkish baths. Studies on Ethno-Medicine 6, 1 – 9. PDF 178. USFDA (US Food and Drug Administration), Online. Poisonous Plant database.

29

Copies of literature reviews

Available at http://www.accessdata.fda.gov/scripts/plantox/index.cfm 179. USDA (US Department of Agriculture), Online. Database for the Flavonoid Content of Selected Foods Release 2.1. Prepared by the Nutrient Data Laboratory, Food Composition Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture. Available at http://www.ars.usda.gov/nutrientdata 180. Van Grutten M, 1980. Carnation dermatitis in a flower seller. Contact Dermatitis 6, p.289. PDF 181. Vidal C and Polo F, 1998. Occupational allergy caused by Dianthus caryophyllus, Gyspophila paniculata and Lilium longiflorum, Allergy 53, 995–998. PDF 182. Wang H, Nair MG, Strasburg GM, Chang CC, Booren AM, Gray JI and DeWitt DL, 1999. Antioxidant and anti-inflammatory activities of anthocyanins and their aglycone, cyanidin, from tart cherries Journal of Natural Products 62, 294-296. PDF 183. Wang H, Conchou L, Bessiere JM, Cazals G, Schatz B and Imbert E, 2013. Flower color polymorphism in Iris lutescens (Iridaceae): biochemical analyses in light of plant- insect interactions. Phytochemistry 94,123-134. PDF 184. WHO, 2001. Anthocyanins. WHO Food additives series 17, Available at http://www.inchem.org/documents/jecfa/jecmono/v17je05.htm. PDF 185. Williamson G, Barroni K, Shimo K and Terao J, 2005. In vitro biological properties of flavonoid conjugates found in vivo. Free Radical Research 39, 457–469. PDF 186. Wu W and Sun R, 2012. Toxicological studies on plant proteins: a review. Journal of Applied Toxicology 32,377-386. PDF 187. Wu X, Pittman HE, McKay S and Prior RL, 2005. Aglycones and sugar moieties alter anthocyanin absorption and metabolism after berry consumption in weanling pigs. The Journal of Nutrition 135, 2417–2424. PDF 188. Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE and Prior R, 2006. Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption. Journal of Agricultural and Food Chemistry 54, 4069-4075. PDF 189. Xie X, Zhao R and Shen GX, 2012. Influence of delphinidin-3-glucoside on oxidized low-density lipoprotein-induced oxidative stress and apoptosis in cultured endothelial cells. Journal of Agricultural and Food Chemistry 60, 1850 – 1856. PDF 190. Yagi M, Kosugi S, Hirakawa H, Ohmiya A, Tanase K, Harada T, Kishimoto K, Nakayama M, Ichimura K, Onozaki T, Yamaguchi H, Sasaki N, Miyahara T, Nishizaki Y, Ozeki Y, Nakamura N, Suzuki T, Tanaka Y, Sato S, Shirasawa K, Isobe S, Miyamura Y, Watanabe A, Nakayama S, Kishida Y, Kohara M and Tabata S, 2013. Sequence analysis of the genome of carnation (Dianthus caryophyllus L.). DNA Research; dst053. PDF 191. Yamagata K, Tagami M and Yamori Y, 2014. Dietary polyphenols regulate endothelial function and prevent cardiovascular disease, Nutrition doi: 10.1016/j.nut.2014.04.011. PDF 192. Yamagishi M, 2013. How genes paint lily flowers: Regulation of colouration and pigmentation patterning. Scientia Horticulturae 163, 27-36. PDF 193. Yang Y, Shi Z, Reheman A, Jin JW, Li C, Wang Y, Andrews MC, Chen P, Zhu G, Ling W and Ni H, 2012. Plant food delphinidin-3-glucoside significantly inhibits platelet activation and thrombosis: novel protective roles against cardiovascular diseases. PLoS ONE 7(5): e37323. doi:10.1371/journal.pone.0037323. PDF 194. Yeh C-T and Yen C-C, 2005. Induction of apoptosis by the anthocyanidins through regulation of Bcl-2 gene and activation of c-Jun N-terminal kinase cascade in hepatoma cells Journal of Agricultural and Food Chemistry 53, 1740–1749. PDF 195. Yu JQ, Yin Y, Lei JC, Zhang XQ, Chen W, Ding CL, Wu S, He XY, Liu YW and Zou GL, 2012. Activation of apoptosis by ethyl acetate fraction of ethanol extract of Dianthus superbus in HepG2 cell line. Cancer Epidemiology 36, e40-45. PDF

30

Copies of literature reviews

196. Yuan Y, Ma X, Shi Y and Tang D, 2013. Isolation and expression analysis of six putative structural genes involved in anthocyanin biosynthesis in Tulipa fosteriana. Scientia Horticulturae 153, 93-102. PDF 197. Yuksel O, Albayrak S, Turk M and Balabanli C, 2010. Utilization possibilities of carnation by-products as an alternative roughage source. Turkish Journal of Field Crops 15, 65 – 68. PDF 198. Yun J-M, Afaq F, Khan N and Mukhtar H, 2009. Delphinidin, an anthocyanidin in pigmented fruits and vegetables, induces apoptosis and cell cycle arrest in human colon cancer HCT116 cells. Molecular Carcinogenesis 48, 260 – 270. PDF 199. Zhang J, Wang L-S, Gao J-M, Xu Y-J, Li L-F and Li C-H, 2012. Anthocyanins from flowers of Viola yedoensis and V. prionantha by high-performance liquid chromatography- photodiode array detection-electrospray ionization mass spectrometry. Phytochemical Analysis 23, 16 – 22. PDF 200. Zhang Y, Vareed SK and Nair MG, 2005. Human tumor cell growth inhibition by nontoxic anthocyanidins, the pigments in fruits and vegetables. Life Sciences 76, 1465- 1472. PDF 201. Zhang Y, Butelli E and Martin C, 2014. Engineering anthocyanin biosynthesis in plants. Current Opinions in Plant Biology 19, 81-90. PDF 202. Zhu M, Zheng X, Shu Q, Li H, Zhong P, Zhang H, Xu Y, Wang L and Wang L, 2012. Relationship between the composition of flavonoids and flower colors variation in tropical water lily (Nymphaea) cultivars. PLoS ONE 7: e34335 doi:10.1371/journal.pone.0034335. PDF 203. Zhu Y, Ling W, Guo H, Song F, Ye Q, Zou T, Li D, Zhang Y, Li G, Xiao Y, Liu F, Li Z, Shi Z and Yang Y, 2013. Anti-inflammatory effect of purified dietary anthocyanin in adults with hypercholesterolemia: a randomized controlled trial. Nutrition, Metabolism, and Cardiovascular Diseases 23, 843-849. PDF 204. Zollner TM, Schmidt P, Kalveram CM, Emman AC and Boechncke W.-H, 2000. Anaphylaxis to red currants. Allergy 55, p.511. PDF

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