vervolg furocoumarine 2

For Key lime pulp, the concentrations of limettin, isopimpinellin and
bergapten were equal; psoralen and xanthotoxin were not detected. Coumarins in lime pulp were 13 to 182 times less concentrated than those in the peel. Based on the amounts and types of coumarins, Persian limes appear to be potentially more phototoxic than Key limes. Although bergapten may be the main component of limes responsible for phytophotodermatitis,
dermatological interaction assays with psoralen, bergapten, xanthotoxin and limettin should be
conducted.

Kaffir lime oil pressed / leaf oil Citrus hystrix DC.
Cropwatch summary Kaffir oil claimed to contain negligible amount of FC’s by some citrus oil manufacturers (private communications to Cropwatch 2007).

Murakami et al. (1999) found the presence of bergamottin, oxypeucedanin & 5-
[(6',7'-dihydroxy-3',7'-dimethyl-2-octenyl)oxy]psoralen in cedrat fruit.

Murakami A., Gao G., Kim O.K., Omura M., Yano M., Ito C., Furukawa H., Jiwajinda S.,
Koshimizu K., & Ohigashi, H. (1999) "Identification of Coumarins from the Fruit of Citrus hystrix
DC as Inhibitors of Nitric Oxide Generation in Mouse Macrophage RAW 264.7 Cells." J. Agric.
Food Chem. 47(1), 333 - 339.

Lemon oils cold pressed - all origins Citrus limon (L.) Burm.
CAS n°: 8008-56-8; EINECS-CAS n°: 84929-31-7.
Cropwatch summary:
Photoxicity. Contact with Tahiti lemons in the S. Brazilian city of Pelotas causes 16 cases of photodermatits per annum: Larangeira de Almeida Jr. & Jorge (undated). Opdyke (1974a) had previously described the phototoxicity of lemon oil, & IFRA currently recommends limit of 2% in fragrances not washed off skin exposed to sunshine. Photo-toxicity of lemon oil cold-pressed is almost totally down to oxypeudedanin & begapten; contents and ratios of these compounds
varies according to origin; average bergapten (range 4-87ppm) & oxypeucedanin (26-728ppm) indicating that the phototoxic potential of oxypeucedanin being a quarter of that of bergapten (Naganuma et al. 1985).
Composition. More specifically Naganuma et al. (1985) deteremined the bergapten content of cold-pressed Ivory Coast lemon oil at 69-87 ppm compared with 5-17ppm for cold-pressed Sicilian lemon oil. Shu et al. (1975) reported the bergapten content of cold-pressed Ivory Coast lemon oil as 11-88 ppm, and for cold-pressed Sicilian lemon oil as 5-17 ppm.
RIFM give the following data “measured by the fragrance industry” (??) for Lemon oil cold-pressed (but no botanical, processing or geographical source details given): psoralen 0-3ppm, bergapten up to 275ppm, isopimpinellin 0- 18ppm, bergamottin up to 5412 ppm, oxypeucedanin up to 8224 ppm; xanthotoxin & angelicin not detected. (RIFM Fact Sheet No 3.).
Earlier data from McHale & Sheridan (1988) for cold-pressed lemon oil had given 2200 ppm bergamottin, 1100 ppm oxypeucedanin, 260 ppm peucedanin hydrate, 180 ppm isoimperatorin, 450 ppm byakangelicol, 70 ppm byakangelicin, 750 ppm 8-geranyloxypsoralen & 60 ppm imperatorin.

Dugo et al. (1998) identified ten furanocoumarins in cold-pressed lemon oil (bergamottin, 8-geranyloxypsoralen, oxypeucedanin, byakangelicol, oxypeucedanin hydrate, byakangelicin, imperatorin, phellopterin, isoimperatorin, 5-isopent-2 -enyloxy-8-(2 ,3 -epoxyisopentyloxy)-psoralen) identifying the main components as bergamottin (160-291 mg/100 g of oil) and 5-geranyloxy-7- methoxycoumarin (180-250 mg/100 g of oil).

Veraza et al. (1999) looked at the effects of the cold-processing method on lemon oil analysis, via looking at the oil characteristics from Pelatrice, Sfumatrice, FMC and Torchi machines. They found that the processing method affects the quantitative composition of the oil (but not the qualitative composition); Pelatrice oils gave the highest amounds of VOC’s but also highest amounts of coumarins & psoralens.

Dugo P., Mondello L., Cogliandro E., Cavazza A.& Dugo G. (2000) "On the genuineness of citrus
essential oils. Part LIII. Determination of the composition of the oxygen heterocyclic fraction of
lemon essential oils (Citrus limon (L.) Burm. F.) by normal-phase high performance liquid
chromatography" Flav & Frag J 13(5), 329-334.

Glueck V. (2006) “Argentina’s lemon industry” Perf & Flav. 31 (6), June 2006 pp22-26. Quote:
“Bergaptene content of single-fold lemon oil (cold-pressed) varied between 0.3048 and 0.3428% for Argentinian lemon oils.”

Naganuma M., Hirose S., Nakayama Y., Nakajima K. & Someya T. (1985) "A study of the
phototoxicity of lemon oil." Arch Dermatol Res. 278(1), 31-6.
Abstract. Lemon oil contains furocoumarin derivatives and is known to cause phototoxicity. In this study, lemon oil was fractionated, and its phototoxic activity was measured by means of a
biological assay. The substances producing phototoxicity were identified by high-performance
liquid chromatography as being oxypeucedanin and bergapten. The phototoxic potency of
oxypeucedanin was only one-quarter of that of bergapten. However, the amounts of these two
phototoxic compounds present in lemon oils produced in different regions of the world varied by a factor of more than 20 (bergapten, 4-87 ppm; oxypeucedanin, 26-728 ppm), and their ratio was not constant. The two compounds accounted for essentially all of the phototoxic activity of all lemon-oil samples. Among various other citrus-essential oils investigated, lime oil and bitterorange oil also contained large amounts of oxypeucedanin. Oxypeucedanin was found to elicit photopigmentation on colored-guinea-pig skin without preceding visible erythema.

Veraza A., Dugo P., Mondello L., Trozzia A. & Cotroneo A. (1999) “Extraction technology & lemon
oil composition.” Italian J Food Science 11(4), 361-370. Abstract. The influence of the extraction
technology on cold-pressed lemon essential oil composition was studied. The volatile and oxygen heterocyclic fractions of cold-pressed lemon essential oils were studied by HRGC, HRGC/MS and normal-phase HPLC. The genuine lemon oils were obtained in the 1997/98 season using the following industrial machines: Pelatrice, Sfumatrice, FMC and Torchi. Sixty-four compounds were identified and quantified. Limonene was the main compound. In the oxygen heterocyclic fraction two coumarins (5-geranyloxy7-methoxycoumarin, citropten) and five psoralens (bergamottin, 8-geranyloxypsoralen, oxypeucedanin, byakangelicol, 5-isopent-2'-enyloxy-8-(2',3'- epoxyisopentyloxy) psoralen were identified and quantified. Bergamottin and 5-geranyloxy-7- methoxycoumarin were the main compounds. It was found that the technology does not influence the qualitative composition of the oil but there were quantitative differences. FMC produced an oil with olfactory notes and a quantitative composition similar to Sfumatrice oil, considered the best quality oil. Pelatrice oils gave the highest amount of volatile oxygenated compounds as well as coumarins and psoralens.

Lemon oil distilled.
Cropwatch summary: No data

Lemon oil terpeneless
Cropwatch summary: No data

Lemongrass oils Cymbopogon spp. incl. C. citratus (dc) Stapf. (West Indian or
Guatamalan oil) & C. flexuosis Stapf. (East Indian oil).
CAS n° 8007-02-1; EINECS-CAS n°: 89998-14-1 (West Indian oil)
CAS n° 8007-02-1; EINECS-CAS n°: 91844-92-7 (East Indian oil)
Cropwatch summary: Oil of C. citratus shows slight phototoxic potential (as shown by modified neutral red uptake assay: Nathalie et al. (2006), Dijoux et al. (2006); however E.I. & W.I. lemongrass oils previously found non-photo-toxic by Opdyke (1976).
Other statements: W.I. Lemongrass oil phototoxicity ‘has not been determined’

Lime oil cold-pressed. – W.I.: Key or Mexican lime from Citrus x aurantiifolia
(Christ.) Swingle, & Persian (syn. Tahitian) lime from C. latifolia Tanaka.
CAS n°: 608008-26-2; EINECS-CAS n°: 90063-52-8.
Cropwatch summary.
Photo-toxicity: IFRA (1992) limits lime oil to 0.7% in fragrances not washed off skin exposed to sunshine based on Opdyke (1974b) & its bergapten content as reported in J.A.O.A.C. (1969) 52(4),727.
Composition FC content noted by Pathak (1962). FC content of West Indian lime peel furanocoumarins 334mg/Kg (mainly limettin); 502mg/Kg for Persian limes (mainly 5-MOP & limettin). Then flesh of both varieties contains 5-6mg/Kg FC’s, mainly isopimpinellin (DFG-Senate Comm 2004). Limettin is only 1/200 times as photoactive as bergapten on rabbit skin.
According to the SCC(NF)P ‘cold pressed lime oil’ – type, botanical source & origin not specified (!) - contains 2.5% bergamottin. However Naganuma et al. (1985) indicate oxypeucedanin as the principle FC. Minor FC’s such as oxypeucedaninyl acetals in Key Lime type A or oxypeucedanin methanolate are still in the process of being characterised in processed oils (Feger et al. 2006). Wagstaff (1991) noted lime oil contained 46.7mg/L bergamottin, isopimpinellin & other FC’s.

RIFM give the following data “measured by the fragrance industry” (??) for Lime oil cold-pressed (but no botanical or geographical source details given): psoralen < 5ppm, bergapten up to 2200 ppm, xanthotoxin < 5ppm, isopimpinellin 2000 ppm, bergamottin 25,000 ppm, oxypeucedanin 1200 ppm; angelicin not detected (RIFM Fact Sheet No 3.).
Refering to McHales data (McHale & Shendan 1989), Frérot & Decorzant (2004) suggest that in the case of cold-pressed lime oils, for example, individual marker compounds such as bergapten may not present constant ratios to the total furanocoumarins on a concentration basis. This is shown below in tabular form.

For Key Limes:  Bergapten ppm               Σ of ten FC’s ppm              Bergapten /Σ   FC’s %
Origin 
Dominica          2000                                  31,800                               6.29
Mexico             2400                                  32,840                               7.3
Peru                1200                                   32,330                              3.71
Haiti                1800                                   29.540                               6.09
Table 1. Key Lime oil data (McHale & Sheriden 1989)

For Persian      Bergapten ppm                Σ of ten FC’s ppm               Bergapten /  Σ   FC’s %
Limes: Origin
Mexico             1500                                 18,850                               7.96
Brazil               1400                                 203,390                             6.87
Florida             2200                                 21,980                               10.01
Table 2. Persian Lime oil data (McHale & Sheriden 1989)

McHale & Sheridan (1989) also produced the following data for the furanocoumarins content of Key lime oil against Persian lime oil.

Furanocoumarin                             Key Lime oil                                 Persian Lime oil
Bergamottin                                      1500-1600                                        1360-1420
Bergapten                                         120-240                                            140-220
Byakangelicol                                     66-100                                              8-16
Oxypeucedanin                                  210-240                                            120-210
Isobyakangelicol                                270-460                                            5-9
Isopimpinellin                                     300-570                                            100-210
8-geranyloxypsoralen                        160-350                                            58-100
Table 3. Furanocoumarins in Key lime oil vs. Persian lime oil (McHale & Sheridan 1989).

Coumarin                  Key Lime Oil A                Key Lime Oil B                   Persian Lime Oil
Citropen                        581                                     484                                     443
Bergamottin                  3408                                   3154                                   3067
Bergapten                     113                                     89                                       217
Oxypeucedanin              - - -                                    144                                      272
Cnidilin                          31                                       24                                        71
Isopimpinellin                336                                     331                                      217
Table 4. Coumarins in various cold-pressed lime oils (Dugo et al. 1997)

DFG-Senate Commission on Food Safety (SKLM) (2004) “Toxicological assessment of
suranocoumarins in food.”

Dugo P., Mondello L., Lamonica G. & Dugo G. (1997) “Characteristics of cold-pressed Key &
Persian lime oils by gas chromatography, gas chromatography/mass spectroscopy, high
performance liquid chromatography & phsiochemical indices.”J. Agric. Food Chem 45, 3608-
3616.

Feger W, Brandauer H, Gabris P. & Ziegler H. (2006) "Nonvolatiles of commercial lime and
grapefruit oils separated by high-speed countercurrent chromatography." J Agric Food Chem. 22, 54(6), 2242-52. Abstract. The nonvolatile fractions of cold-pressed peel oils of Key and Persian lime as well as grapefruit were separated by high-speed countercurrent chromatography (HSCCC).
In addition to the isolation of the main coumarins, psoralens and polymethoxyflavones, a
number of minor constituents were enriched and successfully characterized by GC-MS and
HPLC-UV. 5,7,8-Trimethoxycoumarin and the cyclical acetals of oxypeucedanin hydrate with
citral were determined as new nonvolatile trace constituents of lime oils and confirmed by NMR
spectroscopy. The citral oxypeucedaninyl acetals were found particularly in Key lime oil type A,
which as a result of the juice-oil contact, is exposed to acidic conditions during industrial
processing. Some of the confirmed minor constituents, such as pabulenol, isooxypeucedanin,
and oxypeucedanin methanolate in lime as well as auraptenol in grapefruit, may have been
generated by hydrolysis-sensitive precursors during CCC separation or their respective industrial processing techniques.

Nigg H.N., Nordby H.E., Beier R.C. , Dillman A., Macias C. & Hansen R.C. (1993) "Phototoxic
coumarins in limes" Food and Chemical Toxicology 31(5), 331-335. Abstract: Coumarins in the
rind and pulp of Persian and Key limes were quantified. In the rind of Persian limes, coumarin
concentrations were in the order: limettin > bergapten > isopimpinellin > xanthotoxin > psoralen.
In the rind of Key limes, psoralen and xanthotoxin were analytically absent; limettin was 10 times more concentrated than either bergapten or isopimpinellin, which were equal in concentration.
Coumarin content in Persian lime pulp was in the order; isopimpinellin > limettin > bergapten >
xanthotoxin > psoralen. For Key lime pulp, the concentrations of limettin, isopimpinellin and
bergapten were equal; psoralen and xanthotoxin were not detected. Coumarins in lime pulp were 13 to 182 times less concentrated than those in the peel. Based on the amounts and types of coumarins, Persian limes appear to be potentially more phototoxic than Key limes. Although bergapten may be the main component of limes responsible for phytophotodermatitis,
dermatological interaction assays with psoralen, bergapten, xanthotoxin and limettin should be
conducted.

Wagner A.M., Wu J.J., Hansen R.C., Nigg H.N. & Beiere R.C. (2002) “Bullous
phytophotodermatitis associated with high natural concentrations of furanocoumarins in limes.” J Contact Dermat. 13(1), 10-4. Abstract: Phytophotodermatitis is a phototoxic reaction, occurring in skin exposed to sunlight after contact with plants containing furanocoumarins. Typical reactions are mild, showing erythema with post-inflammatory hyperpigmentation. A 6-year-old boy presented with marked, symmetric, painful erythema and edema of both hands that rapidly developed into dramatic bullae covering the entire dorsum of the hands. The history revealed that the hands had been bathed in lime juice for a prolonged period in the preparation of limeade.
OBJECTIVE: This report documents an unusual bullous presentation of phytophotodermatitis
resulting from contact with furanocoumarins in local limes. This study was conducted to identify
and measure the inciting substances from the rind and pulp of the limes. METHODS: Psoralen,
xanthotoxin, bergapten, and isopimpinellin content were measured by gas chromatography and
high-pressure liquid chromatography RESULTS: The rind contained 6- to 182-fold concentrations
of all furanocoumarins measured when compared with pulp. Bergapten was the most abundant
substance in the rind. CONCLUSION: Hydration of the skin during the preparation of limeade
combined with increased levels of bergapten in local limes to produce a dramatic bullous
reaction. We encourage clinicians to consider the possibility of phytophotodermatitis in severe
bullous skin reactions

Lime oil distilled.
CAS n°: 8008-26-2; EINECS-CAS n°: 90063-52-8
Cropwatch summary: Distilled lime oil is not photo-toxic (Tisserand & Balacs (1995) p147, quoting Opdyke 1974).

Opdyke D.L.J. (1974) Food Cosm.Toxicol 12, 723.

Tisserrand R. & Balacs T. (1995) Essential Oil Safety for Health Professionals Churchill
Livingstone.

Lime oil terpeneless.
Cropwatch summary: No data

Limette peel oil Citrus limetta Risso
Cropwatch summary: No data
Buiarelli F, Cartoni G, Coccioli F, Jasionowska R, Mazzarino M. (2002) "Analysis of limette and
bergamot distilled essential oils by HPLC." Ann Chim. 92(4), 363-72. Abstract. This work
examines the distilled essential oils of limette and bergamot in order to assess the presence of
low volatile substances such as coumarins (bergapten) which, being toxic, must be eliminated
before using these oils in the food industry. The quantitative determination of coumarins was
carried out by spectrofluorimetric detection. The substances present in the chromatograms,
obtained by HPLC with UV detection at 254 nm, were then identified. Moreover, a new coumarin
that is present in small quantities was identified using HPLC-MS.

Lovage oil Levisticum officinale Koch.
CAS n°: 8016-31-7; EINECS-CAS n°: 84837-06-9
Cropwatch summary: Plant leaves contain bergapten; FC concentration
diminishes after flowering (Ojala 2001).

Ashwood Smith M.J., Ceska O., Yeoman A. & Kenny P.G.W. (1992) "Photosensitivity from
harvesting lovage (Levisticum officinale)." Contact Dermatitis 26, 365-357

Mandarin oil - cold pressed Citrus reticulata Blanco.
CAS n°: 708008-31-9; EINECS-CAS n° 84929-38-4.
Cropwatch summary: The oxygenated hetrocyclic compounds in mandarin oil are mainly polymethoxyflavones. Cold-pressed mandarin oil contains 250 ppm bergapten: according to IFRA. Conversley, RIFM gives the following data “measured by the fragrance industry” (??) for mandarin oil (but no botanical or geographical source details given): bergapten 0-3 ppm, bergamottin 0-10 ppm. Psoralen, xanthotoxin, isopimpernellin, oxypeucedanin & angelicin not detected (RIFM Fact Sheet No 3.).

Mandarin petitgrain oil (syn. Petitgrain mandarinier).
CAS n°: 8014-17-3; EINECS-CAS n°: 84929-38-4
Cropwatch Summary: IFRA advises that typical 5-MOP content of petitgrain mandarin oil is 50 ppm (but no botanical or geographical source details given).
Murraya koenigii Spreng. (the Curry Plant).
Seeds contain xanthotoxin, isobyakangelicol, phellopterin, gosferol, neobyakangelicol, byakangelicol, byakangelicin and isogosferol (Adebajo & Reish (2000).

Adebajo A.C. & Reisch J. (2000) "Minor furocoumarins of Murraya koenigii" Fitoterapia 71(3),334-
337.

Myrrh oil. Botanical sources: Commiphora myrrha (Nees) Engl. var. molmol
(Fam. Burseraceae) & other Commiphora spp. such as C. abyssinica (Berg. )
Engl., C. schimperi (Berg.) Engl. Still other source species include C. africana, C.
mucul and C. guidotti.
CAS n°: 8016-37-3; EINECS-CAS No: 9000-45-7
Cropwatch Summary: Not phototoxic in spite of substituted coumarins content.

Neroli oil Citrus x aurantium L. flowers.
CAS n°: 8016-38-4; EINECS CAS n°: 72968-50-4.
Cropwatch summary: phototoxic reactions to neroli oil reported historically (Greenberg & Leicester 1954 - through BoDD).

Greenberg L.A. & Lester D. (1954) Handbook of Cosmetic Materials. Interscience NY.

Opoponax qualities. Botanical sources: Commiphora erythraea (Ehrenb.) Engl.
syn. Commiphora erythraea Engl. var. glabrescrens Engl. & from C. kataf (Forsk.) Engl. C. guidottii & C. holtziana.
Cropwatch summary: Opoponax qualities are a rich source of furano- and dihydrofuranocoumarins, but are not photo-toxic. Roots however contain imperatorin (Appendino et al. 2004). IFRA limits opoponax concentration in fragranced products to 0.6% on sensitization grounds.

Appendino G, Bianchi F, Bader A, Campagnuolo C, Fattorusso E, Taglialatela-Scafati O, Blanco-
Molina M, Macho A, Fiebich BL, Bremner P, Heinrich M, Ballero M, Muñoz E. (2004) “Coumarins
from Opopanax chironium. New dihydrofuranocoumarins and differential induction of apoptosis by imperatorin and heraclenin.” J Nat Prod. 67(4):532-6

Orange oil, bitter – cold pressed Citrus aurantium L. subsp. amara L.
EINECS-CAS n° 72968-50-4
Cropwatch summary:
Phototoxicity:
Based on RIFM work, an IFRA limit of 1.25% for products not washed off the skin was based on an uncertainty factor imposed over data of Kaidbey & Kligman (1980).
Composition: Seville orange flesh contains 13mg/Kg furanocoumarins, & Seville orange marmalade contains 2mg/Kg furanocoumarins (MAFF 1993).
Orange oil bitter contains ‘large amounts’ of oxypeucedanin (Naganuma et al. 1985); Dugo et al. (1996) determined the presence of the coumarins osthol, meranzin, isomeranzin, and meranzin hydrate as well as the furanocoumarins bergapten, epoxybergamottin, and epoxybergamottin hydrate), and four polymethoxyflavones (tangeretin, 3,3',4',5,6,7,8-heptamethoxyflavone, nobiletin, and tetra-O-methylscutellarein) were identified.

RIFM give the following data “measured by the fragrance industry” for orange oil bitter (but no botanical, process or geographical source details given): psoralen 70 ppm. Bergapten 350 ppm, bergamottin 5 ppm, epoxybergamottin 620 ppm (RIFM Fact Sheet No 3). This contrasts with the data from Frerot & Decorzant (2004) obtained by UV-DAD from cold-pressed bitter orange oil C. aurantiium subsp amara: bergapten 1671.1 ppm, oxypeucedanin 457.8 ppm,
epoxybergamottin 814.2 ppm, 8-geranyloxypsoralen 119.0, bergamottin 111.6 ppm.

Buiarelli F, Cartoni GP, Coccioli F, Leone T. (1996) "Analysis of bitter essential oils from orange
and grapefruit by high-performance liquid chromatography with microbore columns." J
Chromatogr A. 730(1-2), 9-16.
Abstract. The analysis of bitter orange and grapefruit essential oils (non-volatile fraction) was carried out by HPLC in normal- and reversed-phase mode with UV detection. These oils were compared with the sweet orange and mandarin essential oils, analyzed previously. For the identification of chromatographic peaks, fractionation by RP-HPLC was carried out. The purified fractions were analyzed by GC-MS and LC-MS. Some new compounds were found, together with many others already identified in different citrus essential oils.

Dugo P., Mondello L., Cogliandro E., Verzera A. & Dugo G. (1996) "On the genuineness of citrus
essential oils. 51. Oxygen heterocyclic compounds of bitter orange oil (Citrus aurantium L.)." J.
Agric. Food Chem., 44 (2), 544 -549, 1996.
Abstract: The composition of the oxygen heterocyclic fraction of bitter orange essential oil, obtained by normal-phase HPLC with two on-line coupled columns (-Porasil and Zorbax), is reported. Genuine, industrial, cold-pressed Italian and Spanish essential oils, commercial oils, laboratory hand-extracted oils, and mixtures of bitter orange oil with sweet orange, lemon, lime, and grapefruit oils were analyzed. Four coumarins (osthol, meranzin, isomeranzin, and meranzin hydrate), three psoralens (bergapten, epoxybergamottin, and epoxybergamottin hydrate), and four polymethoxyflavones (tangeretin, 3,3',4',5,6,7,8- heptamethoxyflavone, nobiletin, and tetra-O-methylscutellarein) were identified. In addition, three unknown coumarins were found. Meranzin was the main component isolated. Meranzin hydrate, the formation of which was probably due to the hydration of meranzin during the industrial extraction, was not found in the laboratory hand-extracted samples. Meranzin was not present in some industrial samples, probably because of a prolonged and unusual contact of the essential oil with an acid aqueous medium during the process of extraction. Italian essential oils usually exhibited a higher content of oxygen heterocyclic compounds than the Spanish oils. The bitter orange oils adulterated by sweet orange oil were characterized by a lower content of almost all the aforementioned components; the adulteration of bitter orange oils with lemon, lime, or grapefruit oils was detected by the presence of components peculiar to added oils.

Fugh-Berman A. & Myers A. (2004) "Citrus aurantium, an Ingredient of dietary supplements
marketed for weight loss: current status of clinical and basic research." Experimental Biology and Medicine 229, 698-704. Quote: C. aurantium contains 6',7'-dihydroxybergamottin and bergapten: Kaidbey, K.H. & Kligman, A.M. (1980). “Identification of contact photosensitizers by human assay.” Current Concepts in Cutaneous Toxicity, 55-68. Academic Press, NY. Report No 1995.
MAFF UK (1993) “Occurrence of linear furocoumarins in the UK diet.”. Joint Food Safety and
Standards Group, Food Surveillance Information Sheet. - see
http://archive.food.gov.uk/maff/archive/food/infsheet/1993/no09/09furo.htm.

Orange oil sweet – cold pressed Citrus sinensis (L.) Osbeck.
CAS n° 8008-57-9; EINECS-CAS n°: 8028-48-6
Cropwatch summary:
Phototoxicity: Nathalie et al. (2006) concluded orange oil sweet was ‘probably phototoxic’ (as shown by modified Sudan Red uptake assay), & peels are known to contain bergapten. On the other hand orange oil sweet is not phototoxic, & not restricted, according to the IFRA Standard.
Composition: The main oxygen heterocyclic components in orange oil sweet appear to be almost entirely composed of polymethoxyflavones, such as tangeritin, heptamethoxyflavone, nobiletin, tetra-O-methylscutellarein, 3,3’,4’,5,6,7- hexamethoxyflavone & sinensetin. (Dugo et al. 1996) [No authors listed]. “Outbreak of severe dermatitis among orange pickers--California.” MMWR Morb Mortal Wkly Rep. (1986 Jul 25) 35(29), 465-7.

Dugo P. Mondello L., Cogliandro E., Verzera A. & Dugo G. (1996) “On the genuineness of citrus
essential oils. 51. Oxygen heterocyclic compounds of bitter orange oil (Citrus aurantium L.)” J.
Agric. Food Chem. 44, 544-549.

Nathalie D., Yannik G., Caroline B., Sandrine D., Claude F., Corrine C., Pierre-Jacques F. (2006)
“Assessment of the Phototoxic Hazard of Some Essential Oils using Modified 3T3 Neutral Red
Uptake Assay.” Toxicol in Vitro 20(4), 480-489.

Orange oil sweet folded Citrus sinensis (L.) Osbeck
Cropwatch summary: No data

Orange oil sweet terpeneless Citrus sinensis (L.) Osbeck
Cropwatch summary: No data

Oxypeucedanin.
Occurs in lemon oil (26 to 728 ppm) and in large amounts in lime & bitter orange oils (Naganuma et al.1985). Photo-toxic potency of oxypeucedanin said to be a quarter of that of bergapten (Naganuma et al.1985). Found at 18.86 ppm by UVDAD in cold expressed Californian lemon oil Citrus limonum (L.) (N. L. Burman) (Frerot & Decorzant 2004). Found at 457.8 ppm in cold-pressed bitter orange oil (Frerot & Decorzant 2004). Dugo et al. (1997) did not detect oxypeucedanin in Key Lime oil A, but found it in Key Lime oil type B & Persian lime oil. The authors speculate that because of the extraction technology used to prepare Key Lime oil
A, where the oil comes in contact with the lime juice, the epoxy-ring opens under these conditions & becomes hydrated to oxypeucedanin hydrate. Oxypeucedanin also occurs in traditional Chinese medicinal herb Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook – see Wei & Ito (2006). According to Naganuma et al. (1985) oxypeucedanin elicited photopigmentation on colored-guinea-pig skin without preceding visible erythema.

A RIFM communiqué FR 08/15 distributed Mar. 2008 indicated that “the studies proposed for oxypeucedanin (CAS# 737-52-0) will not be conducted due to the inability to find a supplier that can provide the volume of test article needed that is not cost-prohibitive.” Cropwatch comments: It is beyond belief that amongst the large number of scientists represented within the RIFM organisation, and its huge operating budget, coupled with IFRA’s boast that it represents 90% of the fragrance trade, that not one chemist could be found who was able to prepare some high purity oxypeucedanin for phototoxicity testing.

Chaudhary S.K., Ceska O., Tetu C., Warrington P.J., Ashwood-Smith M.T., Poulton G.A.. (1986).
“Oxypeucedanin, a major furanocoumarin in parsley, Petroselinum crispum.” Planta Medica 6,
462-464.
Dugo P., Mondello L., Lamonica G. & Dugo G. (1997) "Characterization of Cold-Pressed Key and
Persian Lime Oils by Gas Chromatography, Gas Chromatography/Mass Spectroscopy, High-
Performance Liquid Chromatography, and Physicochemical Indices" J. Agric. Food Chem. 45(9),
3608 -3616, 1997
Abstract. The physicochemical indices and the qualitative and quantitative composition of the volatile fraction and the oxygenated heterocyclic fraction of cold-pressed Key lime oil (types A and B) and Persian lime oil are reported. The volatile fraction of Persian lime oil is characterized by a higher content of limonene, g-terpinene, esters, and monoterpene aldehydes and a lower content of b-pinene + sabinene, sesquiterpenes, and aliphatic aldehydes than Key lime oils. Oxypeucedanin was not detected in Key lime oil type A, while it is present in Key lime oil type B and Persian lime oil. This is probably due to the extraction technology used for Key lime oil type A, which allows the essential oil to come into contact with the juice. Under these conditions, the epoxy ring of oxypeucedanin is opened by hydrolysis to form oxypeucedanin hydrate.
Frérot E. & Decorzant E. (2004) "Quantification of total furocoumarins in citrus oils by HPLC
coupled with UV, fluorescence and mass detection." J Agr. Food Chem. 52: 6879-6886.

Naganuma M., Hirose S., Nakayama Y., Nakajima K. & Someya T. (1985) "A study of the
phototoxicity of lemon oil." Arch Dermatol Res. 278(1), 31-6. Abstract. Lemon oil contains
furocoumarin derivatives and is known to cause phototoxicity. In this study, lemon oil was
fractionated, and its phototoxic activity was measured by means of a biological assay. The
substances producing phototoxicity were identified by high-performance liquid chromatography as being oxypeucedanin and bergapten. The phototoxic potency of oxypeucedanin was only onequarter of that of bergapten. However, the amounts of these two phototoxic compounds present in lemon oils produced in different regions of the world varied by a factor of more than 20 (bergapten, 4–87 ppm; oxypeucedanin, 26–728 ppm), and their ratio was not constant. The two compounds accounted for essentially all of the phototoxic activity of all lemon-oil samples. Among various other citrus-essential oils investigated, lime oil and bitter-orange oil also contained large amounts of oxypeucedanin. Oxypeucedanin was found to elicit photopigmentation on coloredguinea- pig skin without preceding visible erythema.

Yun W. & Ito Y. (2006) "Isolation of imperatorin, Oxypeucedanin, and isoimperatorin from
Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook by Stepwise Flow Rate High‐Speed
Countercurrent Chromatography." Journal of Liquid Chromatography & Related Technologies
29(11), 1609-1618.

 Oxypeucedanin hydrate
Found at 131.91 ppm by UV-DAD in cold expressed Californian lemon oil Citrus limonum (L.) (N. L. Burman) (Frerot & Decorzant 2004). Oxypeucdanin hydrate formed from oxypeucedanin (see above) in Key lime oil A can further interact with citral produces a series of cyclical acetals which are particularly characteristic of this oil (Feger et al. 2006).

Feger W, Brandauer H, Gabris P, Ziegler H. (2006) "Nonvolatiles of commercial lime and
grapefruit oils separated by high-speed countercurrent chromatography." J Agric Food Chem.
54(6), 2242-52.
Abstract. The nonvolatile fractions of cold-pressed peel oils of Key and Persian lime as well as grapefruit were separated by high-speed countercurrent chromatography (HSCCC).
In addition to the isolation of the main coumarins, psoralens and polymethoxyflavones, a
number of minor constituents were enriched and successfully characterized by GC-MS and
HPLC-UV. 5,7,8-Trimethoxycoumarin and the cyclical acetals of oxypeucedanin hydrate with
citral were determined as new nonvolatile trace constituents of lime oils and confirmed by NMR
spectroscopy. The citral oxypeucedaninyl acetals were found particularly in Key lime oil type A,
which as a result of the juice-oil contact, is exposed to acidic conditions during industrial
processing. Some of the confirmed minor constituents, such as pabulenol, isooxypeucedanin,
and oxypeucedanin methanolate in lime as well as auraptenol in grapefruit, may have been
generated by hydrolysis-sensitive precursors during CCC separation or their respective industrial processing techniques.

Parsley herb/seed oils/oleoresin Petroselinium crispum (Miller) A.W. Hill.
CAS n°: 8008-68-8; EINECS CAS n°: 84012-33-9
Cropwatch summary: Toxicity associated with furanocoumarin content (Newall et al. 1996). IFRA advises that typical 5-MOP content of parsley leaf oil is 20 ppm. Contrastingly, parsely (fresh leaves & roots) said to contain 70-100 ppm oxypeucedanin, & the furanocoumarins psoralen, bergapten, xanthotoxin, isoimperatorin, isopinpinellin & gravelone (Chaudhary et al. 1986). Myristicin, a cancer chemopreventative agent, is also component of parsley leaf oil, is an
inducer of the detoxifying glutathione S-transferase (GST) in mouse tissue
(Zheng et al. 1992).

Afek U., Orenstein J., & Aharoni N. (2002) “The involvement of marmesin and its interaction with
GA3 and psoralens in parsley decay resistance” Can. J. Plant Pathol. 24, 61–64 (2002) Quote: “It is known that +marmesin is the precursor of psoralens (linear furanocoumarins) in species
belonging to the families of Umbelliferae, Apiaceae, Rutaceae, Moraceae, and Leguminoseae)”
Jahnen W. & Hahlbrock K. (1987) "Differential regulation and tissue-specific distribution of
enzymes of phenylpropanoid pathways in developing parsley seedlings." Planta Med 173(4),
453-458. Quote "Isopimpinellin & xanthotoxin or bergapten or both detected in exudates from oil ducts of plants.”

Zheng G.Q., Kenney P.M., Zhang J., & Lam L.K. (1992) "Inhibition of benzo[a]pyrene-induced
tumorigenesis by myristicin, a volatile aroma constituent of parsley leaf oil." Carcinogenesis.
13(10), 1921-3.

Parsnip extracts / oil Pastinaca sativa L. var. sativa hortensis Erh.
CAS n°: 68917-22-6; EINECS-CAS n°: 90082-39-6
Cropwatch summary: Tissues contain bergaptene, xanthotoxin, angelicin, sphondin, isopimpinellin, imperatorin. Exact FC levels of seed/herb/root oils unknown to Cropwatch, but FC’s are not destroyed in parsnip roots by cooking (Ivie et al. 1981). Parsnip oil, which contains several octyl esters, is used in trace amounts as a modifier in perfumery. Also used in alcoholic beverage flavourings, reportedly an ingredient in Schnapps (Burfield 2000).
Beattie P.E., Wilkie M.J., Smith G., Ferguson J. & Ibbotson S.H. (2007) “Can dietary
furanocoumarin ingestion enhance the erythemal response during high-dose UVA1 therapy? J
Am Acad Dermatol. 56(1):84-7. Abstract. As phototoxic skin reactions caused by psoralen are
induced by wavelengths within the UVA1 spectrum, we assessed the potential of the small
amount of psoralen in a normal diet to provoke phototoxicity in volunteers with skin types I and II. Threshold erythema was unaffected by ingestion of a 200-g portion of parsnip.
Berembaun M.R., Zangerl A.R., Nitao J.K. (1984). “Furanocoumarins in seeds of wild and
cultivated parsnip.” Phytochemistry 23, 1809-1810.

Burfield T. (2000) Natural Aromatic Materials – Odours & Origins. Ist edn published AIA, Tampa
Ivie G., Holt D. & Ivey (1981) “Natural toxicants in human foods psoralens oin raw & cooked
parsnip root.” Science 213, 909-910

Lutchman L, Inyang V, Hodgkinson D. (1999) “Phytophotodermatitis associated with parsnip
picking.” J Accid Emerg Med. 16(6), 453-4.

Petitgrain bigarade oil C. aurantium / C. aurantium v. aurantium leaves.
CAS No: 8016-44-2; EINECS-CAS n° 72968-50-4.
Cropwatch summary: Phototoxic reactions to oil reported (Greenberg & Leicester 1954, Klarmann 1958 – through BoDD). Conversely, petitgrain oil was not found phototoxic: RIFM monograph FCT 20, 801 Sp Issue VI.
In the real world, the other petitgrain oils (mandarinier, citronnier, bergamottier), are often of variable composition (i.e. blends of several petitgrain leaf oils). IFRA advises that typical 5-MOP content of Petitgrain mandarin oil is 50 ppm.

Greenberg L.A. & Lester D. (1954) Handbook of Cosmetic Materials. Interscience NY.
Klarmann E.G. (1958) “Perfume Dermatitis” Ann. Allergy 16, 425.

Petitgrain bigarade sur fleurs d’oranger. [C. aurantium essential oil distilled
over C.aurantium flowers]
Cropwatch summary; No data:

Photo-allergy.
Is, according to Spielmann et al. (2000) “an acquired immunological reactivity, which does not occur on first, treatment with a photosensitiser, and light /UV radiation, and needs an induction period of one or two weeks before skin reactivity can be demonstrated by administration of photosensitiser and irradiation with light/UV radiation.” It is therefore a delayed type of
hypersensitivity which involves the binding of a photosensitiser chemical to skin protein(s).

Kaidbey K.H. & Kligman A.M. (1980) "Photomaximization test for identifying photoallergic contact
sensitizers." Contact Dermatitis. 6(3), 161-9. Abstract. The photomaximization procedure was
designed to identify topical photocontact sensitizers following the format of the maximization test for contact sensitizers. The test agent is applied for 24 hours followed by exposure to three
Minimal Erythema Doses (MED) of solar simulated radiation twice weekly for 3 weeks (six
exposures) in a panel of 25 white Caucasoids. The subjects are challenged 2 weeks later with 4.0 J/cm2 of long-wave ultraviolet radiation (UV-A). Photocontact sensitization was induced to
3,3'4',5-tetrachlorosalicylanilide (TCSA); dibromosalicylanilide (DBS) but not to tribomosalicylanilide unless the latter was contaminated with DBS. Jadit and bithionol were weak photoallergens. The highest rate of sensitization was given by 6-methylcoumarin, a widely used synthetic fragrance. Hexachlorophene and trichlorocarbanilide were negative.
Ljunggren B. (1977) "Psoralen photoallergy caused by plant contact." Contact Dermatitis 3(2), 85- 90. Abstract. A case of acquired photocontact allergy to furocumarins in plants is reported.
Photopatch testing was performed with four psoralens [8-methoxypsoralen (8-MOP), 5-
methoxypsoralens (5-MOP), trimethylpsoralen (TMP) and imperatorin (IMP)]. The use of serial
dilutions of the test compounds made it possible to differentiate between photoallergic and
phototoxic reactions. 8-MOP gave a positive eczematous test reaction down to a concentration of 0.0001%. The reactions to 5-MOP and imp also were positive, while that to TMP awas negative. Histopathological examination of a biopsy specimen from a positive test site showed changes consistent with photoallergic contact dermatitis. The multiple reactions could be explained on the basis of multiple sensitization but cross reactions cannot be ruled out.
Lovell W.W. & Jones P.A. (2000) "Evaluation of mechanistic in vitro tests for the discrimination of
photoallergic and photoirritant potential." Altern Lab Anim. 28(5), 707-24.
Abstract. Photochemical tests were used to discriminate photoallergens and photoirritants. UV absorption spectrometry was employed to identify chemicals which absorbed sunlight wavelengths and which required further testing. Photoallergic potential was assessed by studying photobinding of the test chemicals to human serum albumin. Photobinding was determined by increased UV absorbance of the protein fraction after gel filtration chromatography. Photooxidation of histidine was used to screen for a mechanism of photoirritancy. Efficient photooxidisers may be considered photoirritant rather than photoallergic. The substances selected for the EU/COLIPA phototoxicity project were
tested. There were 14 photoirritants (three tested as both free acid/base and salts, i.e., 17
samples in total), four photoallergens, three which were photoirritant and photoallergenic (i.e., 17 photoirritants and seven photoallergens) and six "negatives" (four clearly non-phototoxic and two unclear). UV spectrometry showed that 28 of the 30 substances absorbed sunlight significantly and had the potential for adverse photoreaction. Six of seven photoallergens were identified as such by the photobinding assay. Most photoirritants also caused photomodification of protein, but eleven of these photooxidised histidine efficiently and so were classified as photoirritants. Four photoirritants remained falsely predicted as photoallergens. Two photoirritants were negative for both photomodification of protein and for histidine photooxidation. Four chemicals negative in vivo were negative in vitro. The remaining two chemicals could not be classified because of unclear data both in vivo and in vitro. The in vitro test battery, therefore, was useful for the discrimination of photoallergic and photoirritant potential.

Neumann N.J., Blotz A., Wasinska-Kempka G., Rosenbruch M., Lehmann P., Ahr H.J., Vohr
H.W. (2005) "Evaluation of phototoxic and photoallergic potentials of 13 compounds by different
in vitro and in vivo methods." Photochem Photobiol B. 79(1), 25-34.
Abstract. Phototoxic side effects of pharmaceutical and cosmetic products are of increasing concern for patients, dermatologists and the chemical industry. Moreover, the need of new chemicals and drugs puts pressure on pre-clinical test methods for side effects, especially interactive adverse-effects with UV-light. So, the predictive potential of different established test methods, which are used regularly in our departments in order to detect the phototoxic potential of chemicals, were analyzed. Namely the fibroblast 3T3 test, the photo hen's egg test, a guinea pig test for measuring acute photoreactions, and a modified Local Lymph Node Assay, the Integrated Model for the Differentiation of Skin Reactions. Various agents with different photoreactive potential were tested: quinolones like Bay y 3118, ciprofloxacin, enoxacin, lomefloxacin, moxifloxacin, ofloxacin, sparfloxacin, as well as promethazine, chlorpromazine, 8-methoxypsoralen and olaquindox serving as control. Special emphasis was taken to evaluate the capability of the employed test procedures to predict phototoxic side effects in patients. Following our results, both in vitro assays were useful tools to detect photoirritancy while the photoallergic potentials of tested compounds were exclusively detected by an in vivo assay. As long as no in vitro model for photoallergy is available, the UV-IMDS should be considered to evaluate photoallergic properties of a supposed photoreactive agent.

Photo-carcinogenicity.
Can be defined as carcinogenity induced by a combination of chemical & repeated light- or UV radiation-exposure.

Schimmer O, Kühne I. (1990). "Mutagenic compounds in an extract from Rutae Herba (Ruta
graveolens L.). II. UV-A mediated mutagenicity in the green alga Chlamydomonas reinhardtii by
furoquinoline alkaloids and furocoumarins present in a commercial tincture from Rutae Herba."
Mutat Res. 243(1):57-62. Abstract. A commercial tincture prepared from Rutae Herba (Ruta
graveolens L.) exhibited a moderate photomutagenicity in an arginine-requiring mutant strain of
Chlamydomonas reinhardtii. In the tincture some furocoumarins, e.g., bergapten, psoralen,
imperatorin, and 3 furoquinoline alkaloids (dictamnine, gamma-fagarine, skimmianine) were
detected. All compounds revealed photomutagenic properties but their activities were quite
different. Bergapten was the most potent furocoumarin. Dictamnine, the furoquinoline with the
strongest effect, reached only about 10% of the activity of bergapten. Based on the amount of
these compounds in the tincture and their activities we conclude that bergapten is mainly
responsible for the photomutagenicity of the tincture. The lower phototoxicity and
photomutagenicity of the furoquinoline alkaloids may be due to the fact that furoquinolines form
only monoadducts with DNA in the presence of UV-A in contrast to furocoumarins which also
form biadducts.

Schimmer O., Kiefer J. & Paulini H. (1991) "Inhibitory effects of furocoumarins in Salmonella
typhimurium TA98 on the mutagenicity of dictamnine and rutacridone, promutagens from Ruta
graveolens L. " Mutagenesis 6(6), 501-506. Abstract. Eight furocoumarins differing in their basic
structure and substitution pattern (angular, linear, dihydrofuran type) were tested for their ability to reduce the mutagenic potency of dictamnine and rutacridone, two alkaloids present in extracts from Ruta graveolens L. Both compounds need metabolic activation by S9 mix in order to exhibit mutagenicity in Salmonella typhimurium strain TA98. The furocoumarins used in this study did not show any mutagenicity either with or without S9 mix within the dose range tested. However, all the furocoumarins were able to inhibit the mutagenicity induced by dictamnine as well as by rutacridone in a dose-dependent manner. Imperatorin turned out to be the most efficient inhibitor.
The inhibitory effect is probably due to the inactivation of the cytochrome P450 enzyme complex
which prevents the activation of the promutagens. This is indicative of the desmutagenic
character of the furocoumarins. However, there is also some evidence that the reduction of the
mutagenicity induced by dictamnine might be caused to a small extent by a mechanism which
possibly depends on the competition with furocoumarins for the same sites in the DNA molecule.

Photo-clasticity
[This below, is what little Cropwatch knows of RIFM Commissioned research on
the photoclasticity of isopimpinellin & bergamottin]:
RIFM contracted David Kirkland of Covance Ltd. UK to carry out studies on photoclasticity studies
on isopimpinellin & bergamottin on their behalf, using samples obtained from Extrasynthese
(RIFM 2007), and. apparently this work was submitted by RIFM to the SCCP in May 2007. We
would be negligent if we did not point out that the New Jersey based-Covance animal testing
company has been the subject of video-evidenced animal cruelty allegations by PeTA in their
Vienna, Va. establishment (see http://blog.peta.org/archives/2007/04/covance_payspe.php), and similarly by BUAV in their investigations into operations at Muster, Germany in 2004 (see
http://buzzle.com/editorials/1-17-2004-49549.asp). Altogether, Cropwatch regards Covance as a pretty strange choice of a research partner by RIFM, the latter being an organisation representing an industry which should be particularly sensitive about its public image wrt the use of animals in the safety testing of cosmetic ingredients.

Although IFRA/RIFM officials never answer our mail enquiries, when Cropwatch contacted Prof.
Kirkland of Covance UK for details of the photoclasticity contracted work, he was gracious
enough to explain why he had to decline, even though we had politely pointed out this was a
public interest health & safety matter. The RIFM scientific report indicates that Kirkland found that bergamottin provided a photoclastic response (structural chromosome aberrations) significantly above background at all concentrations tested in the presence of UV light, whereas isopimpinellin induced aberrations not significantly different from the controls. Unless RIFM allow Kirkland to publish the work, or the SCCP publishes the entire work itself, then presumably the exact details of this work will remain hidden from public scrutiny.

A RIFM scientific report (RIFM May 2007) revealed that Kirkland found that bergamottin provided
a photoclastic response (structural chromosome aberrations) significantly above background at
all concentrations tested in the presence of UV light, whereas isopimpinellin induced aberrations
not significantly different from the controls.

Photo-clastogenicity – other articles.
Interestingly, photoclastogenicity has also been associated with other cosmetic materials such as zinc oxide & titanium dioxide.

Dufour E.K., Kumaravel T., Nohynek G.J., Kirkland D. & Toutain H. (2006) "Clastogenicity, photoclastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in preirradiated or simultaneously irradiated Chinese hamster ovary cells." Mutat Res. 607(2), 215-24.
Abstract. Zinc oxide (ZnO), a widely used ingredient in dermatological preparations and
sunscreens, is clastogenic in vitro, but not in vivo. Given that ZnO has an approximately four-fold greater clastogenic potency in the presence of UV light when compared with that in the dark, it has been suggested to be photo-clastogenic. In order to clarify whether this increased potency is a genuine photo-genotoxic effect, we investigated the clastogenicity of ZnO (mean particle size, 100 nm) in Chinese hamster ovary (CHO) cells in the dark (D), in pre-irradiated (PI, i.e. UV irradiation of cells followed by treatment with ZnO) and in simultaneously irradiated (SI, i.e. ZnO treatment concurrent with UV irradiation) CHO cells at UV doses of 350 and 700 mJ/cm(2). The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was as follows: SI>PI>D. In the dark, ZnO produced a concentration-related increase in chromosome aberrations (CA). In PI or SI CHO cells, ZnO was clastogenic at significantly lower concentrations
(approximately two- to four-fold) when compared with effective concentrations in the dark, indicating an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV
irradiation per se. The incidence of CA in SI or PI cells was generally higher than that in the dark.
At similar ZnO concentrations, SI conditions generally produced higher CA incidence than PI
conditions. However, when ZnO concentrations producing similar cytotoxicity were compared, CA incidences under PI or SI conditions were nearly identical. The modest increase in the
clastogenic potency of ZnO following UV irradiation contrasts with the results observed with
genuine photo-clastogenic agents, such as 8-MOP, which may produce an increase in
clastogenic potency of >15,000-fold under SI conditions. Our results provide evidence that, under conditions of in vitro photo-clastogenicity tests, UV irradiation of the cellular test system per se may produce a slight increase in the genotoxic potency of compounds that are lastogenic in the dark. In conclusion, our data suggest that minor increases in clastogenic potency under
conditions of photo-genotoxicity testing do not necessarily represent a photo-genotoxic effect, but may occur due to an increased sensitivity of the test system subsequent to UV irradiation.

Theogaraj E, Riley S, Hughes L, Maier M, Kirkland D. (2007) "An investigation of the photoclastogenic potential of ultrafine titanium dioxide particles." Mutat Res. 634(1-2), 205-19.
Abstract. Ultrafine titanium dioxide is widely used in a number of commercial products including
sunscreens and cosmetics. There is extensive evidence on the safety of ultrafine titanium dioxide. However, there are some published studies indicating that some forms at least may be
photogenotoxic, photocatalytic and/or carcinogenic. In order to clarify the conflicting opinions on
the safety of ultrafine titanium dioxide particles, the current studies were performed to investigate the photo-clastogenic potential of eight different classes of ultrafine titanium dioxide particles. The photo-clastogenicity of titanium dioxide was measured in Chinese hamster ovary (CHO) cells in the absence and presence of UV light at a dose of 750 mJ/cm(2). The treatments were short (3 h) followed by a 17-h recovery and achieved concentrations that either induced approximately 50% cytotoxicity or reached 5000 microg/ml if non-cytotoxic. None of the titanium dioxide particles tested induced any increase in chromosomal aberration frequencies either in the absence or presence of UV. These studies show that ultrafine titanium dioxide particles do not exhibit photochemical genotoxicity in the model system used.

Photo-genotoxic / photo-mutagenic & genotoxic effects.
Photogenotoxicity is a genotoxic response observed after exposure to the combination of a photosensitizing chemical, and a non-genotoxic dose of lightor UV- radiation.

Averbeck D. (1985) “Relationship between lesions photoinduced by mono- and bi-functional
furocoumarins in DNA and genotoxic effects in diploid yeast.” Mutation Research/Fundamental
and Molecular Mechanisms of Mutagenesis 151, 217-233.

Averbeck D., Cundari E., Dardalhon M., Dall'Acqua F. & Vedaldi D. (1990) "Genetic effects and
repair of DNA photo-adducts induced by 8-methoxypsoralen and homopsoralen
(pyranocoumarin) in diploid yeast." J Photochem Photobiol B. 5(2), 179-95. Abstract. The
relationship between DNA mono- and di-adducts and genetic effects induced by the
pyranocoumarin 8,8-desmethylxanthyletine (homopsoralen) HP and 365 nm radiation (UVA) was
investigated in the diploid yeast strain D7 (Saccharomyces cerevisiae) taking 8-methoxypsoralen 8-MOP) as a reference compound. The number of DNA cross-links (CLs) induced was determined using alkaline step elution analysis. The induction and removal of total photo-adducts was followed using radioactively labelled compounds. HP showed the same photobinding capacity as 8-MOP. As a function of UVA dose, it was less effective than 8-MOP for the induction of CLs and genetic effects. However, as a function of CLs induced, HP was shown to be more effective for the induction of lethal effects and mitotic recombination than 8-MOP but equally effective for the induction of mutations. The results suggest that, although CLs are recognized as genetically effective lesions, at a given number of CLs, HP induced mono-adducts efficiently contribute to the induction of lethal effects and mitotic recombination but less to the induction of mutations. Using a re-irradiation protocol, HP was brought to yield the same relative amounts of CLs at the same number of total adducts as single UVA exposures with 8-MOP. In these conditions, mutation induction and the kinetics for the removal of photo-adducts were the same for both agents indicating that not only the removal of adducts but also mutation induction are highly dependent on the relative level of CLs induced.

Averbeck D., Averbeck S., Dubertret L., Young A.R., Morlière P. (1990) "Genotoxicity of
bergapten and bergamot oil in Saccharomyces cerevisiae." J Photochem Photobiol B. 7(2-4),
209-29. Abstract. In order to determine the genotoxic potential of bergapten (5-methoxypsoralen (5-MOP] and bergamot oil (BO), the genetic effects of 5-MOP and BO (containing equivalent amounts of 5-MOP) were studied in haploid and diploid yeast (Saccharomyces cerevisiae) using solar simulated radiation (SSR). At equal doses of SSR, equal concentrations of 5-MOP alone or 5-MOP in BO have a similar influence on survival and on the induction of cytoplasmic "petite" mutations, reverse and forward mutations, mitotic gene conversion and genetically aberrant colonies including mitotic crossing over. No reciprocity is found between SSR dose and 5-MOP concentration for cytotoxic, mutagenic and recombinogenic effects. In the presence of chemical filters (Parsol 1789, a UVA filter, and Parsol MCX, a cinnamate derivative acting as a UVB filter) considerable protection is observed against the induction of genetic effects by 5-MOP and BO containing 5-MOP in haploid and diploid cells. As indicated by the lower induction kinetics, the protection is higher than expected from the light-absorbing properties, suggesting photochemical interaction. The protection is slightly higher for BO than for 5-MOP. The induction of genetic effects by 5-MOP alone or BO containing 5-MOP is independent of oxygen. Experiments on suction blister fluids taken from patients after topical treatment with BO containing 5-MOP indicate that in comparison with water the bioavailability and thus the genotoxic effects of the compounds are decreased. Moreover, in addition to the filtering effect against the photoinduced genotoxic effects of BO, the presence of chemical filters apparently reduces the penetration of BO containing 5-MOP and provides a reduction in biological effectiveness.

Chetalat A., Dresp J.H. & Gocke (1993) “Photomutagenesis test development 2. 8-
methoxypsoralen, chlorpromazine & sunscreen compounds in chromosomal aberration assays
using CHO cells. Mutation Research 292, 251-258.
Abstract. Chromosomal changes were analysed in Chinese hamster ovary (CHO) cells treated with 8-methoxypsoralen (8-MOP) or chlorpromazine (CPZ) and irradiated with either a UVA fluorescent tube (emission spectrum ranging from 350 to 400 nm) or a xenon burner (continuous emission spectrum simulating ambient sunlight). In the dark neither 8-MOP nor CPZ was genotoxic by itself. If these compounds were used in combination with UV irradiation the rate of chromosome aberrations was significantly increased. The magnitude of the clastogenic response was dependent on compound concentration and UV dose. The spectral composition also played an important role. Care must be taken to account for spectral changes caused, e.g., by passage of the light through the plastic lid of the container. The possible clastogenicity of two sunscreens was tested with two protocols: (1) cells attached to the culture dish were treated in presence of the sunscreen in the medium or (2) cells were irradiated through a layer of sunscreen solution as a filter. With this a clear UVB-absorbing effect and a decreased frequency of UVAB-induced chromosome aberration was evident with the UVB-absorbing compound Parsol HS but was absent, as expected, with the UVA-absorbing compound Parsol 1789. The presence of the sunscreens in the irradiated cell sample did not cause a significant increase in UV-induced chromosome aberrations.

naar vervolg furocoumarine 3