pirolisis (6% dari tembakau asli) dan 40% dalam pirolisa residu tembakau yang diekstraksi (94%

dari tembakau asli). Schlotzhauer dan Schmeltz (3466) juga menunjukkan dengan pirolisis
komponen individu yang diisolasi dari ekstrak heksana bahwa berikut ini adalah prekursor PAH
dalam pirolisa: n-dotriacontane, asam stearat dan asam linoleat, phytol, squalene, dan
β-sitosterol.

Pada tahun 1973, Chortyk dan Schlotzhauer (722) mengkaji pirogenesis komponen
asap tembakau. Mereka membahas hasil ekstraksi / pirolisis yang dijelaskan sebelumnya
ditambah hasil studi mereka pada pirolisis (800 ° C) dari fraksi yang secara berurutan
diekstraksi dari tembakau dengan serangkaian pelarut yang semakin polar (heksana, aseton,
etanol, air) (3458). Chortyk dan Schlotzhauer (722) mencatat:

Sumber sekitar 70% hidrokarbon aromatik mulai dari benzena hingga BaP, dalam
pirolisa, disebabkan oleh komponen daun yang dapat diekstraksi dengan heksana dan
aseton: ekstrak ini berjumlah kurang dari 25% dari daun kering berat. Ekstrak heksana
(7,2% dari berat daun) menyumbang 33% dari produk netral. Bahan yang larut dalam
heksana dari tembakau daun yang diawetkan diketahui meliputi parafin alifatik dan siklik,
asam lemak, fitosterol, ester steryl, dan terpena, semua prekursor hidrokarbon aromatik
yang disukai. Ekstrak aseton (17,5% dari berat daun) menyumbang 35% hidrokarbon
aromatik lainnya ke pirolisa, menunjukkan ekstraksi lebih lanjut dari prekursor
hidrokarbon aromatik polisiklik aromatik yang serupa. Bersama-sama, kedua ekstrak ini
menyumbang 86% dari kandungan BaP dari pirolisis tembakau. Kontribusi yang tidak
proporsional dari fraksi-fraksi ini terhadap pembentukan BaP menjadi jelas dari data
berikut: fraksi heksana dan aseton, yang mengandung hidrokarbon dan lipid,
menghasilkan sekitar 700 mg [sic] * BaP per gram bahan yang dihidrolisis, sedangkan
tembakau residual, sebagian besar terdiri dari bahan selulosa, menghasilkan kurang
dari 34 mg [sic] * BaP per gram pirolisis.

Mereka juga menunjukkan bahwa fenol volatil utama (fenol, kresol) diproduksi terutama oleh
pirolisis alkohol yang dapat diekstraksi dan residu tembakau akhir. Fraksi-fraksi ini
masing-masing menyumbang 38% dan 44% dari total fenol. Alkohol yang dapat diekstraksi
termasuk pigmen tembakau polifenolik dan gula dengan berat molekul rendah, sedangkan
residu tembakau akhir mengandung selulosa polisakarida, pati, pektin, dan lignin. Semua ini
menghasilkan fenol sederhana pada pirolisis atau selama merokok tembakau (248, 2043, 3277,
3305, 3453, 3468, 3767). Pada 1978, Severson et al. (3616) mengunjungi kembali ekstraksi
pelarut organik tembakau. Mereka mencatat [lihat hal. 277 in (3616)]:

Sejumlah studi pirolitik telah menunjukkan bahwa ekstrak heksana atau minyak bumi ... ekstrak
tembakau mengandung prekursor utama hidrokarbon aromatik poliklik asap [Wynder et al.
(4355), Schlotzhauer et al. (3468), Schlotzhauer dan Chortyk (3451)]. Namun, banyak dari
penelitian ini dilakukan dalam kondisi yang menghasilkan hasil PAH optimal [Schmeltz dan
Hoffmann (3489)]; dengan demikian, distribusi PAH tidak sebanding dengan yang ada di [CSC]
dan data yang diperoleh tidak dapat dikorelasikan secara tepat dengan produksi PAH selama
proses merokok.
Severson et al. (3616) menentukan kondisi pirolitik yang memberikan profil PAH dari pirolisis
tembakau yang mereka klaim "dapat dikorelasikan dengan profil PAH [CSC]." Pemeriksaan
data mereka menunjukkan bahwa klaim ini tidak valid. Severson et al. melakukan dua
percobaan besar. Mereka memeriksa:

1. Pengaruh ekstraksi petroleum eter pada tingkat PAH dalam pirolisa dari petroleum
eter yang dapat diekstrak PEE (8% dari berat tembakau asli), residu tembakau yang
diekstraksi (92% dari berat tembakau asli), dan tembakau asli.
2. Pengaruh kromatografi dari petroleum eter yang dapat diekstraksi PEE dengan
delapan sistem pelarut yang semakin polar, mulai dari petroleum eter hingga aseton:
larutan metanol, pada komposisi masing-masing dari delapan fraksi kromatografi dan
pada tingkat PAH dalam pirolisa dari masing-masing dari delapan fraksi.

Tabel 25.3, diadaptasi dari (3616), menunjukkan bahwa, pada kondisi pirolisis optimum yang
ditentukan (700 ° C, N2), total dari kelompok PAH (kecuali untuk kelompok benzopyrene) dalam
pirolisa dari petroleum eter extractables (PEE) dan residu tembakau yang diekstraksi (RES)
jauh lebih tinggi (42% -89%) daripada tingkat kelompok PAH yang sama dalam pirolisis dari
tembakau yang tidak diekstraksi. Tabel 25.3 juga menunjukkan bahwa jumlah total PAH dalam
pirolisis dari dua fraksi [PEE dan residu tembakau yang diekstraksi (RES)] adalah 61% lebih
besar dari total PAH dalam pirolisis tembakau kontrol. Studi serupa pada asam individu dan
total serta fenol individu dan total dalam pirolisa yang sama mengungkapkan bahwa jumlah total
fenol dalam pirolisa PEE dan total fenol dalam residu tembakau yang diekstraksi (RES) adalah
21% lebih sedikit daripada total fenol dalam kontrol pirolisis tembakau; untuk asam, jumlah total
asam dalam pirolisa dari dua fraksi (PEE ditambah residu) adalah 13% lebih besar dari total
asam dalam pirolisa tembakau kontrol. Jika situasi ini berlaku dalam situasi yang relatif
sederhana (PEE, residu tembakau yang diekstraksi, dan tembakau kontrol yang tidak
diekstraksi) yang dijelaskan sebelumnya, maka sangat mungkin bahwa perbedaan akan
meningkat ketika senyawa individu dipertimbangkan, yaitu, akan ada ketidakpastian substansial
dalam setiap berupaya mengekstrapolasi dari data pirolisis pada senyawa tertentu, baik yang
ada di dalam atau yang diusulkan untuk ditambahkan ke tembakau, ke apa yang sebenarnya
akan terjadi pada senyawa tersebut selama merokok. Sejak Severson et al. (3616)
mendefinisikan komposisi dari delapan fraksi kromatografi, sisanya dari temuan penelitian
mereka akan dibahas pada bagian berikut.

25.3 KOMPONEN TEMBAKAU

INDIVIDU

Penelitian yang dilakukan dari awal 1950-an hingga pertengahan1960-an tentang

prekursor organik yang larut dalam pelarut dalam tembakau PAH dalam rokok MSS telah
dijelaskan secara terperinci oleh Wynder dan Hoffmann [lihat hal. 345–351, 518 dalam (4332) )]
dan Hoffmann dan Wynder (1798). Terlepas dari kenyataan bahwa PAH tumorigenik pada level
mereka dalam asap rokok dan bertindak bersamaan dengan mempromosikan dan / atau PAH
cocarcinogenic tetapi nontumorigenic, fenol, asam, dll., Pada level mereka dalam asap rokok
hanya menjelaskan sebagian kecil (kurang dari 2%). ) dari respon biologis (diukur sebagai%
TBA) yang diamati pada hewan laboratorium yang dicat dengan kulit, upaya untuk
mendefinisikan prekursor utama dalam tembakau dari PAH tumorigenik dalam asap rokok terus
berlanjut hingga akhir tahun 1970-an, terutama oleh kelompok USDA di Athens, GA (3616).
Terlepas dari kemajuan selama periode ini dalam teknik laboratorium untuk (1) fraksinasi
campuran kompleks yang berhasil seperti yang ditemukan dalam SPM rokok dan dalam pirolisa
dari berbagai tembakau, fraksi tembakau, dan komponen tembakau dan (2) kuantisasi tingkat
PAH individu yang teridentifikasi, fenol, aldehida, keton, dll., tidak ada prekursor baru yang
revolusioner dalam tembakau PAH dalam asap tembakau yang ditemukan: Komponen
tembakau yang larut dalam pelarut organik yang menghasilkan PAH pada pirolisis pada
dasarnya adalah yang atau secara struktural mirip dengan yang telah ada. mengusulkan
beberapa dekade sebelumnya pada 1950-an. Beberapa komponen tembakau, yang secara
struktural mirip dengan yang diusulkan sebelumnya tidak diidentifikasi sebagai komponen
tembakau pada 1950-an, tetapi begitu mereka diidentifikasi, logika mendikte mereka akan
menghasilkan PAH baik pada pirolisis atau selama proses merokok tembakau. Misalnya, pada
akhir 1950-an, Wright [(4282), lih. Rodgman (3243)] berteori bahwa terpena dengan berat
molekul tinggi (selain pitosterol) dan pitosterol sendiri merupakan prekursor utama dalam
tembakau dari PAH dalam asap tembakau. Proposal ini kemudian dikonfirmasi: Pada tahun
1958, Rodgman dan Cook (3269, 3291) menunjukkan dalam percobaan "spiking" dengan
solanesol bahwa itu adalah prekursor yang efektif dari PAH total dan individu dalam
SPM rokok sebagaimana ditunjukkan oleh peningkatan kadar PAH di SPM dari rokok tembakau
solanesol- “berduri” vs SPM dari rokok kontrol tembakau. Demikian pula, Rodgman dan Cook
melaporkan bahwa hasil dari rokok tembakau phytosterol- "berduri" menunjukkan bahwa
pitosterol adalah prekursor utama PAH di MSS. Pada tahun 1979, Severson et al. (3616)
menunjukkan bahwa pirolisis solanesol menghasilkan sejumlah besar PAH vs komponen
tembakau organik terlarut-pelarut lainnya.

Pada bagian selanjutnya, studi pirolisis pada berbagai komponen tembakau yang dikategorikan
sebagai berikut akan dibahas:

Nikotin komponen organik yang larut dalam pelarut: rantai panjang hidrokarbon alifatik jenuh
dan tak jenuh berantai panjang; alkohol terpenoid seperti duvanediol, pitosterol, piton, dan
solanesol; alkohol alifatik berantai panjang normal; ester dari gugus alkohol ini dengan asam
alifatik rantai panjang (palmitat, stearat, oleat, dll.)

Komponen struktural tembakau (biopolimer seperti lignin dan polisakarida selulosa, pati, dan
pektin dan konstituennya monosakarida seperti glukosa dan fruktosa). )

Asam Protein dan

asam amino

Nikotin (dan juga alkaloid yang berhubungan dengan nikotin lainnya dalam tembakau, biasanya
terdapat dalam jumlah yang sangat sedikit) adalah komponen tembakau yang kadarnya dalam
tembakau kadang-kadang dikontrol oleh penghilangan dalam proses denikotinisasi. Berbeda
dengan penghilangan atau pengurangan levelnya dalam kasus nikotin, bahan-bahan seperti
gula sederhana, gliserol, dan beberapa zat penyedap ditambahkan ke campuran tembakau
untuk menambah level mereka yang ada dalam tembakau dan untuk meningkatkan sifat-sifat
organoleptik tertentu yang dapat diterima oleh konsumen. SPM. Bahan-bahan seperti licorice,
kakao, dan zat penyedap lainnya ditambahkan untuk memberikan sifat organoleptik lain yang
dapat diterima oleh konsumen terhadap asap (2341).

25.3.1
NicotiNe

Selama bertahun-tahun, hasil berbagai penelitian tentang pirolisis nikotin telah dipublikasikan.
Banyak dari mereka, misalnya, dari Frank et al. (25A22) dan Woodward et al. (4275a, 25A84,
25A85) pada awal 1940-an, mendahului eskalasi pada tahun 1950-an dari minat terhadap
masalah kesehatan asap rokok. Studi awal lain dari pirolisis nikotin di udara atau nitrogen
termasuk yang di awal 1960-an oleh Jarboe dan Rosene (1923a) dan Kobashi et al. (25A37,
25A38). Pada tahun 1964, produk pirolisis yang dihasilkan dari nikotin selama proses merokok
tembakau dirangkum oleh Kuhn (2228). Pada tahun 1960, Van Duuren et al. (4027)
memperluas studi mereka pada PAH tumorigenik dalam CSC arus utama untuk
mengidentifikasi tiga senyawa N-heterosiklik tumorigenik: aza-arena dibenz [a, h] acridine,
dibenz [a, j] acridine, dan 7H-dibenzo [c, g] -carbazole. Hasil mereka masing-masing 0,1, 2,7,
dan 0,7 ng / cig, secara substansial kurang dari hasil ng / cig yang dilaporkan untuk B [a] P
pada awal 1960-an. Ketiga N-heterosiklik ini juga telah dilaporkan sebagai tumorigen kulit-tikus
[lihat tabulasi dalam Hartwell (1544)]. Dibenz [a, h] acridine dan dibenz [a, j] acridine juga
diidentifikasi oleh Van Duuren et al. dalam pirolisa dari nikotin dan piridin (750 ° C, N2). Namun,
keberadaan bakteri ini dalam asap tembakau atau pirolisis nikotin telah dipertanyakan. Dari
banyak laporan tentang golongan senyawa ini dalam asap tembakau, hanya satu laporan yang
menjelaskan identifikasi salah satu dari ketiganya, dibenz [a, j] acridine: Wynder dan Hoffmann
(4319, 4332) melaporkan bahwa kelompok mereka, Candeli et al. (587), dapat mengkonfirmasi
keberadaan dibenz [a, j] acridine tetapi tidak dibenz [a, h] acridine di CSC mainstream.
Hasil per rokok untuk dibenz [a, j] acridine dilaporkan oleh Candeli et al. (587) adalah hampir
empat kali lipat yang dilaporkan oleh Van Duuren et al. Sejak 1963, tidak ada peneliti lain yang
melaporkan ketiga senyawa ini dalam asap tembakau atau pirolisis nikotin. Pada tahun 1970,
Kaburaki et al. (2006) melaporkan hasil pirolisis nikotin mereka pada berbagai suhu di udara
dan di N2. Dua benzacridine tumorigenik dilaporkan dalam pirolisis nikotin oleh Van Duuren et
al. karena komponen pirolisa nikotinnya tidak ditemukan oleh Kaburaki et al. Schmeltz et al.
(3499) menggambarkan hasil pirolisis mereka dari beberapa komponen tembakau nitrogen,
termasuk nikotin:

Kami tidak dapat mendeteksi benzo (a) piren dalam pirolisis nikotin, juga tidak dapat kami
mengkonfirmasi keberadaan dibenzacridine yang aktif secara fisiologis dan dibenzcarbazole
yang dilaporkan dalam asap tembakau dan dalam pirolisis nikotin dan piridin oleh Van Duuren
[4027].

Dalam ulasan mereka tentang pirogenesis komponen asap tembakau, Chortyk dan
Schlotzhauer (722) menekankan kegagalan beberapa peneliti untuk mengkonfirmasi dua
dibenzacridine dan 7H-dibenzo [c, g] karbazol dalam nikotin pirolisis:

Karena nikotin adalah yang paling berlimpah dan terbaik alkaloid tembakau yang dikenal,
pirolisisnya telah dipelajari secara menyeluruh [Woodward et al. (4275a), Jarboe dan Rosene
(1923a)]. Pekerjaan yang lebih baru [Kaburaki et al. (2006)] tentang pirolisis nikotin dan
berbagai alkylpyridine telah menghasilkan mekanisme yang diusulkan untuk degradasi termal
nikotin ... Schmeltz [Schmeltz et al., (3499)] juga mempelajari nikotin dan mengidentifikasi
sejumlah senyawa yang sebelumnya tidak dilaporkan di pirolisis nikotin ... Ini termasuk pirol,
acenaphthene, indole, skatole, dan antrasena dan / atau fenantrena. Namun, keberadaan
dibenzacridine dan dibenzcarbazole, yang sebelumnya dilaporkan dalam nikotin dan pyridine
pyrolysates, tidak dapat dikonfirmasi [Van Duuren et al. (4027)].

Pada tahun 1977, Schmeltz dan Hoffmann (3491) dalam ulasan mereka tentang komponen
tembakau dan asap tembakau yang mengandung N membahas pembentukan berbagai piridin
dari nikotin selama proses merokok sebenarnya dan pirolisis. Schmeltz dan Hoffmann (3491)
melaporkan identifikasi oleh Van Duuren et al. (4027) dari dua dibenzacridine dalam asap rokok
dan pirolysate nikotin. Namun, mereka tidak mengomentari ketidakmampuan peneliti lain (587,
2006, 3499) untuk mengkonfirmasi temuan Van Duuren et al. Pada tahun 1979, Schmeltz et al.
(3512) melaporkan temuan utama mereka dari studi tentang nasib nikotin radiolabeled selama
pirolisis dan selama merokok aktual dari rokok yang diobati dengan nikotin radiolabel:

Di bawah kondisi pirolisis tabung pembakaran, nikotin dalam matriks gel silika (suhu pirolisis =
600 ° C , 750 ° C, atau 900 ° C) atau matriks tembakau (600 ° C) mengalami degradasi yang
luas menjadi piridin, kuinolin, arilnitril, dan hidrokarbon aromatik. Dalam rokok yang terbakar
selama perokok sebenarnya, sebagian besar nikotin (sekitar 41%) tetap utuh, 12,5% dioksidasi
menjadi karbon dioksida, sebanyak 11% terdegradasi menjadi alkilpiridin yang mudah
menguap, dan jumlah yang dapat diabaikan dikonversi menjadi netral atau asam. komponen
fase partikulat. Dibenz [a, h] acridine dan dibenz [a, j] acridine dilaporkan hampir dua dekade
sebelumnya oleh Van Duuren et al. (4027) tidak diidentifikasi dalam penelitian ini.

Schmeltz et al. (3512)

mencatat:

Dalam penelitian yang sedang berlangsung kami sekarang mengidentifikasi

senyawa-senyawa yang terbentuk dari nikotin hanya sebagai senyawa minor (<0,1%)
yang tetap dapat berkontribusi terhadap toksisitas asap. Untuk kelompok konstituen
asap kecil ini memiliki nikotin sebagai prekursor milik dibenzacridine.
Dari hasil percobaan mereka, Schmeltz et al. (3512) menyimpulkan bahwa percobaan pirolisis
mungkin memiliki nilai terbatas untuk menentukan nasib nikotin dan komponen tembakau
lainnya dalam rokok yang terbakar. Sistem pirolisis dalam penelitian ini [lih. Higman et al.
(1648)] dirancang untuk menjadi simulasi optimal dari proses merokok. Jelas, nikotin tidak
berperilaku dalam sistem pirolisis ini seperti yang terjadi pada rokok yang terbakar selama
merokok. Pemeriksaan 1979 Schmeltz et al. publikasi tentang nasib nikotin selama pirolisis vs
merokok aktual mengungkapkan setidaknya dua penulis (Hoffmann, Schmeltz) pasti mengubah
pendapat mereka tentang pandangan lama mereka tentang kesetaraan perilaku majemuk
selama pirolisis dan merokok aktual, misalnya, Wynder dan Hoffmann (4332) sebelumnya
menulis:

Kebanyakan studi pirolisis dengan tembakau, ekstrak tembakau, fraksi ekstrak, komponen
individu, dan aditif tembakau dilakukan dalam atmosfer nitrogen. Prosedur ini telah sering
dikritik dengan alasan bahwa banyak konstituen beracun yang terbentuk selama merokok
produk tembakau terjadi sebagai akibat dari pembakaran di udara daripada di atmosfer
nitrogen. Kritik ini, bagaimanapun, tidak dapat dipertahankan dalam pandangan studi oleh
Newsome dan Keith [2780] yang menunjukkan bahwa ada pengurangan daripada suasana
pengoksidasi ada di daerah kerucut rokok yang terbakar.

Berkenaan dengan dibenzacridine dan dibenzocarbazole dari nikotin selama proses pirolisis
dan merokok, Tabel 25.4 merangkum tingkat pengetahuan: Tidak ada investigasi yang
dilakukan dari tahun 1963 hingga 2000 pada tingkat ketiga aza-arena di CSC menegaskan
temuan 1960 dari Van Duuren et al. (4027). Aspek lain dari keterlibatan nikotin (dan alkaloid
tembakau terkait) dalam pembentukan komponen tembakau dan / atau asap yang diduga
berbahaya adalah pembentukan N-nitrosamin (TSNAs) khusus tembakau. * Diklaim oleh
Hoffmann et al. bahwa TSNAs seperti N′-nitrosonornicotine (NNN) dan 4- (N-methylnitrosamino)
-1- (3-pyridyl) -1butanone (NNK) dibentuk dari nikotin dan alkaloid kecil terkait nikotin lainnya
selama berbagai tahap pengembangan tembakau dan pengobatan (tumbuh, menyembuhkan,
penuaan) [Hecht et al. (1563, 1564), Adams et al. (22)]. TSNA muncul dalam MSS rokok
sebagai hasil dari dua mekanisme: (1) transfer TSNA langsung dari tembakau ke asap, (2)
pirogenesis selama proses merokok. Misalnya, Hoffmann et al. (1734) dan Adams et al. (29)
memperkirakan bahwa 40% -46% dari NNN dalam MSS adalah hasil transfernya dari campuran
tembakau. Sisanya — 54% -60% — dalam MSS disebabkan oleh pirogenesis NNN dari nikotin
dan nornikotin selama proses merokok. Data serupa dihasilkan untuk NNK: 26% -37% dengan
transfer langsung, 63% -74% dengan pirogenesis. Tingkat MSS dari TSNAs seperti NNN
mungkin dapat dikurangi dengan pengurangan tingkat nikotin dan / atau nitrat dalam campuran
tembakau (499). Namun, Brunnemann et al. (511) telah menyajikan data yang bertentangan
tentang pentingnya korelasi antara kadar nitrat, nikotin, dan TSNA dalam tembakau. Karena
TSNA adalah komponen dari fase partikulat MSS, kontrol level mereka dalam asap juga
dimungkinkan dengan cara apa pun yang mengontrol hasil dari partikel MSS. Ini termasuk yang
berikut:

• Penggunaan lembar tembakau

yang dilarutkan
• Tembakau yang
diperluas
• Efisiensi penyaringan
yang tinggi
• Pengenceran udara dengan meningkatnya porositas kertas dan / atau tip filter
 berventilasi [Hoffmann et al. (1719)]

Berbeda dengan N-nitrosamin yang mudah menguap seperti dimethylnitrosamine (DMNA),

TSNA tidak dapat menerima penyaringan selektif dengan menggunakan ujung filter yang
diplastikkan.
25.3.2 KOMPONEN Larut Larut ORGANIK (Hidrokarbon alifatik berantai panjang,
fitosterois, Solanesol, ester berbobot molekul tinggi, dll.)

Komponen asap tembakau “dikenal” pada tahun 1954 berjumlah kurang dari 100 (2170).
Pada waktu itu, komposisi kimia tembakau sama tidak jelasnya dengan asapnya. Pirolisis yang
dilakukan sepanjang tahun 1950 melibatkan bahan yang larut dalam pelarut organik dari
tembakau dan terbatas baik pada total yang dapat diekstraksi atau untuk beberapa kelas
komponen tembakau yang diketahui, misalnya, pitosterol, hidrokarbon alifatik jenuh berantai
panjang. Senyawa yang berat molekulnya relatif tinggi ini hadir dalam tembakau pada tingkat
yang biasanya melebihi 0,5% dan menimbulkan sedikit masalah dalam mengumpulkan bahan
yang cukup untuk dipelajari. Penelitian selanjutnya pada komposisi tembakau (dan asap
tembakau) menghasilkan isolasi dan penjelasan struktur berbagai kelas senyawa organik yang
larut dalam pelarut, termasuk yang tercantum dalam Tabel 25.5. Karya awal Roffo (3327)
tentang pengurangan tumorigenisitas dari "destilat destruktif" dari tembakau yang diekstraksi
dengan pelarut (etil alkohol), klaimnya bahwa ekstraksi alkohol menghilangkan prekursor
tumorigen dari tembakau, dan usulannya tentang pitosterol sebagai prekursor tumorigen telah
dijelaskan sebelumnya. Terlepas dari kenyataan bahwa "distilat destruktif" dari tembakau tidak
setara dengan masalah partikulat asap rokok, usulan pitosterol sebagai prekursor tembakau
PAH didasarkan pada alasan yang masuk akal dan temuan eksperimental sebelumnya. Hampir
dua dekade sebelumnya, Kennaway (2073-2075) telah menunjukkan bahwa pirolisis isoprena,
asetilena, dan kolesterol, menghasilkan pirolisis tumorigenik pada kulit tikus. Kolesterol pernah
dianggap unik untuk jaringan hewan, tetapi kemudian, jumlah jejak ditemukan dalam jaringan
tanaman, termasuk tembakau [Stedman (3797), Grunwald et al. (1434)]. Dari tembakau dan
sterol asap tembakau, dua yang hadir pada tingkat tertinggi adalah stigmasterol dan
β-sitosterol. Ini berbeda satu sama lain dan kolesterol dalam struktur dan konfigurasi rantai
samping pada cincin cyclopentane. Mereka muncul dalam tembakau sebagai fitosterol bebas
dan turunan fitosteril (ester, glikosida). Pirolisis sterol menghasilkan tingkat tinggi chrysene.
Keempat cincinnya secara konfigurasi mirip dengan sistem cincin sterol. Pada pertengahan
1920-an, Kennaway dan yang lainnya prihatin dengan nasib kolesterol ketika jaringan hewan
dipanaskan selama memanggang dan memanggang. Percobaan pirolisis kolesterol adalah
upaya mentah untuk menduplikasi apa yang terjadi padanya selama proses memasak. Di sini
sekali lagi, keberadaan komponen jaringan lain (asam amino, protein, lipid, karbohidrat) dengan
kolesterol selama memasak tentu akan mempengaruhi hasil jika dibandingkan dengan hasil
yang diperoleh dari pirolisis kolesterol saja. Pada tahun 1928, Kennaway dan Sampson (2080)
juga menunjukkan bahwa tumorigenicities spesifik pirolisa dari kolesterol dan senyawa organik
lainnya meningkat ketika suhu pirolisis meningkat. Pengamatan ini kemudian dikonfirmasi
dengan hidrokarbon alifatik jenuh (2257) dan fitosterol [Wynder et al. (4355, 4356)] diisolasi dari
tembakau. Dalam dua studi terakhir, hasil PAH yang dihasilkan selama pirolisis senyawa
tersebut meningkat ketika suhu pirolisis meningkat. Wynder et al. (4355) dan Wynder dan
Hoffmann (4332) melaporkan bahwa pirolisis dari ekstrak heksana tembakau memberikan hasil
yang menurun dari "tar" pirolisis ketika suhu pirolisis meningkat: 560 ° C, 50%; 640 ° C, 35%;
720 ° C, 32%; 800 ° C, 28%; dan 880 ° C, 28%. Mereka juga melaporkan bahwa pirolisis pada
880 ° C dari heksana yang dapat diekstraksi di udara vs pirolisis mereka di N2 memberikan
pirolisa yang hasilnya pada dasarnya sama [30% (udara) vs 28% (N2)] dan yang memiliki
tumorigenicities spesifik dapat dibandingkan di studi melukis kulit dengan tikus dan kelinci.
Temuan serupa dilaporkan untuk hidrokarbon tembakau alifatik yang dirolisis baik di udara atau
di N2 pada 800 ° C sehubungan dengan komposisi PAH dan tumorigenicity spesifik dalam studi
pengecatan kulit. Tabel 25.6, diadaptasi dari Lam (2257), menunjukkan hubungan antara
produksi PAH dan suhu pirolisis untuk hidrokarbon tembakau alifatik yang dirolisis di udara
pada beberapa suhu. Perhitungan rasio hasil [PAH, mg / g / B [a] P, mg / g] dari PAH lain vs B
[a] P mengungkapkan informasi yang signifikan: Dalam kasus
pirolisis ini, tidak ada konsistensi antara perubahan dalam rasio PAH / B [a] P karena suhu
meningkat dari 700 ° C menjadi 800 ° C, misalnya, dalam kasus PAH tetrasiklik, rasio PAH / B
[a] P menurun untuk piren dan krisan tetapi meningkat untuk fluoranthene; untuk PAH
pentasiklik, rasio menurun untuk perilen dan B [e] P; untuk PAH hexacyclic dibenzo [def, mno]
chrysene, rasionya meningkat. Tren yang sama ini ada apakah rasio PAH / B [a] P dihitung
sebagai hasil molar atau, seperti pada Tabel 25.6, sebagai jumlah absolut (μg PAH yang
dihasilkan per gram hidrokarbon tembakau alifatik pirolisis). Signifikansi dari data dan
perhitungan ini adalah demonstrasi mereka pada tahun 1956 bahwa dalam situasi pirolisis yang
paling sederhana sekalipun, B [a] P bukanlah "indikator" yang valid untuk PAH dengan empat
cincin atau lebih dan hubungan mereka dengan aktivitas tumorigenik [Wynder dan Hoffmann
(4317, 4319, 4332)]. Selain data ini oleh LAM, data sebaliknya lainnya yang menunjukkan
ketidakabsahan konsep B [a] P sebagai "indikator" untuk PAH dengan empat cincin atau lebih
dan tumorigenisitas substrat (CSC, pirolisis) yang mengandungnya adalah dihasilkan tidak
hanya oleh Wynder et al. (4355, 4356) tetapi juga oleh Campbell dan Lindsey (583), Rodgman
dan Cook (3286), Gori (1329, 1330, 1332, 1333), NCI (2685), dan Severson et al. (3616).
Kurangnya korelasi antara konten CSC B [a] P dan tumorigenicity spesifik ditunjukkan oleh
Lazar et al. (2320) yang melaporkan bahwa peningkatan 30 kali lipat dalam konten B [a] P
dengan penambahannya ke CSC tidak menghasilkan peningkatan tumorigenisitas spesifik
untuk kulit tikus dari B [a] CSC yang ditingkatkan P vs kontrol CSC yang diterapkan pada
tingkat dosis yang sama. Kurangnya korelasi antara konsentrasi B [a] P dalam CSC dan
tumorigenisitas spesifiknya juga diakui oleh US Surgeon General yang menulis 1981 [lihat hal.
36 in (4009)]:

Kontribusi BaP atau PAH secara umum terhadap karsinogenesis kulit tikus oleh
kondensat asap rokok tidak dapat sepenuhnya diukur pada saat ini. Wynder dan
Hoffmann [4332] menemukan korelasi antara kadar BaP dan aktivitas karsinogenik dari
kondensat asap dari beberapa jenis rokok. Serangkaian rokok eksperimental yang jauh
lebih besar dipelajari dalam program merokok dan kesehatan di National Cancer
Institute. Tidak ada ketergantungan signifikan dari potensi karsinogenik pada konten
BaP yang diamati [Gori (1329, 1330, 1332, 1333), NCI (2683)].
Stimulus pasca-1930 untuk penelitian PAH disediakan oleh peristiwa-peristiwa berikut: sintesis
independen DB [a, h] A pada tahun 1929 oleh CLAR (760) dan Fieser dan Dietz (1184);
demonstrasi tumorigenisitas kulit tikusnya oleh Kennaway dan Hieger (2078); laporan awal
1930-an oleh Cook et al. (726, 727) pada beberapa isolat PAH dari tar batubara, yang dikenal
bersifat tumorigenik pada kulit tikus dan kelinci; identifikasi dua isolat tar batubara sebagai B [a]
P dan B [a] A; dan demonstrasi tumorigenisitas pada kulit tikus B [a] P (194, 726). Selain
Kennaway dan Sampson (2080), banyak peneliti setelah 1932/1933 memeriksa pirolisis dari
sterol dalam hubungannya tidak hanya dengan dugaan tumorigenisitas dan kandungan PAH
dari "distilat destruktif" dari tembakau, CSC, dan pirolisa tembakau, ekstrak tembakau, dan
masing-masing komponen tembakau tetapi juga dengan dugaan tumorigenisitas dan
kandungan PAH dari bahan makanan yang dipanaskan dan perannya dalam kanker saluran
pencernaan. Sebagai contoh, Roffo, di samping studi tumorigenisitasnya dengan "distilat
destruktif" dari berbagai jenis tembakau (3320, 3324, 25A51) dan tembakau organik yang
diekstraksi dengan pelarut (3327), menyelidiki tumorigenisitas lemak dipanaskan atau
teroksidasi (25A53, 25A55, 25A59) dan "ter" dan turunan fenantrena dari kolesterol pirolisis
(25A56, 25A57, 25A58) atau diiradiasi di udara (25A54). Peneliti lain yang memeriksa sifat
kimia dan biologis dari pirolisis kolesterol termasuk Steiner et al. (25A69), Falk et al. (1171), dan
Bischoff dan Rupp (25A10). Sebelumnya, Veldstra (4042a) telah menunjukkan bahwa
3,5-kolestadien, diproduksi secara pirolitik dari kolesterol, adalah tumorigenik untuk hewan
laboratorium yang dicat kulit. Selanjutnya, cholesten-4-one ditemukan bersifat tumorigenik
untuk kulit tikus dan komponen pirolisa dari kolesterol dan / atau turunannya. Ini dan penemuan
pirolisa lainnya pada komposisi (PAH dalam pirolisa) dan sifat-sifat
(tumorigenicity dari pirolisa dan / atau komponen pirolisa yang dihasilkan dari kolesterol atau
turunannya yang alami) dirangkum oleh Rodgman (3233, 3242). Walaupun PAH
1,2-dihydro-3-methylbenz [j] aceanthrylene pentacyclic (sebelumnya dikenal sebagai
3-methylcholanthrene atau 20-methylcholanthrene), tumorigen kulit-tikus yang kuat, dapat
dibuat dengan serangkaian reaksi kimia dari sterol- senyawa turunan yang secara struktural
terkait dengan kolesterol, tidak pernah diidentifikasi dalam sterol pirolisis. Meskipun Kröller
(2191) mengklaim identifikasi 1,2-dihydro-3methylbenz [j] aceanthrylene (3-methylcholanthrene)
dalam CSC arus utama, identifikasinya itu dipertanyakan oleh Wynder dan Hoffmann (4332)
yang menyatakan bahwa itu dan PAH yang teralkilasi lainnya belum pernah dilaporkan sebagai
produk pembakaran atau pirolisis. Wynder dan Hoffmann (4332) juga mempertanyakan laporan
oleh Pietzsch (2962) dan Kröller (2191) tentang kehadiran di CSC dari PAH DMB yang
termetilasi [a] A. Ini dan PAH termetilasi lainnya dalam CSC arus utama dilaporkan pada tahun
1960 oleh Rodgman dan Cook (3273). Selain itu, pada tahun 1963, Grossman et al. (1431,
1432) melaporkan naftalena teralkilasi dalam pirolisa dari solanesol, komponen tembakau
utama. Pada 1970-an, Snook et al. melaporkan tidak hanya beberapa dimethylbenz [a]
antrasena di CSC (3756) tetapi juga sejumlah besar PAH teralkilasi di CSC (3757). Dengan
demikian, pernyataan Wynder-Hoffmann tentang keberadaan PAH teralkilasi dalam MSS tidak
benar. Kolesterol, stigmasterol, β-sitosterol, dan kolesterol telah diidentifikasi bebas dan / atau
terikat (sebagai ester, dll.) Dalam tembakau dan CSC [lihat Grunwald et al. (1434) untuk
ringkasan penelitian awal tentang sterol tembakau dan turunannya]. Dalam studi mereka,
Grunwald et al. menemukan bahwa keempat sterol ini membentuk sekitar 0,16% dari berat
tembakau, dan sekitar 15% di antaranya dipindahkan ke SPM. Menurut Grunwald et al., Sisa
sterol adalah “Hilang dalam sidestream asap, pirolisis selama proses merokok dan / atau
disimpan di pantat.” Dengan demikian, sebatang rokok berisi 1,0 g tembakau yang dipelajari
oleh Grunwald et al. akan mengandung 1600 μg sterol ini dan mengirimkan sekitar 240 μg ke
MSS. Sekitar 82 ug dari 240 ug akan menjadi β-sitosterol, suatu senyawa yang dilaporkan
bersifat anti kanker terhadap beberapa N-nitrosamin [Grunwald et al. (1434)]. Ini menunjukkan
proporsi relatif dari empat sterol ini dalam tembakau: Kolesterol adalah yang paling banyak dari
empat sterol tembakau pada Tabel 25.7. Kecuali untuk perbedaan kecil dalam struktur rantai
samping, cholesteryl oleate yang dipelajari oleh Veldstra (4042a) secara struktural mirip dengan
fraksi ester fitosteril yang diisolasi dari tembakau oleh Rowland dan Latimer (3358) dan dari
CSC oleh Rodgman et al. (3296). Kemudian diidentifikasi sebagai campuran ester stigmasterol
dan β-sitosterol dengan asam jenuh (palmitat, stearat) dan asam tak jenuh (oleat, linoleat)
berantai panjang. Pada akhir 1950-an hingga awal 1960-an, Rodgman dan Cook tidak berhasil
dalam studi CSC mereka untuk mengidentifikasi diena yang berasal stigmasterol atau
β-sitosterol atau keton yang sesuai dengan tumorigenik 3,5-kolestadien dan kolesten-4-satu
yang dihasilkan selama pirolisis dari kolesterol atau esternya. However, Benner et al. (273)
subsequently identified 3,5-campestadiene (VIb) and 3,5-stigmastadiene (VId) in tobacco
smoke [cf. Eatough et al. (1099, 1100)]. The following paragraphs summarize the relationships,
both known and proposed, between a sterol such as cholesterol and its various pyrolysis
products: Because of the results of the studies by Kennaway (2073– 2076), Kennaway and
Sampson (2080), and Roffo (25A56, 25A57, 25A58) on the generation of tumorigenic
pyrolysates from cholesterol or cholesterol-containing foodstuffs, the question was raised: What
is the relationship between these observations on tumorigenic cholesterol pyrolysates and the
incidence of stomach and digestive tract cancers? When many studies, beginning in 1934 [see
summary in Hartwell (1544)], revealed that 1,2-dihydro-3- methylbenz[j]aceanthrylene
(3-methylcholanthrene) was a potent tumorigen equivalent in potency in mouse-skin-painting
bioassays to B[a]P and DB[a,h]A, extensive research was conducted in attempts to determine
whether it was generated from cholesterol during various cooking processes. Examination of the
entries in the catalogs by Hartwell (1544) and Shubik and Hartwell (3664) for the four PAHs
considered
to be potent tumorigens revealed 474 studies involving 1,2-dihydro-3-methylbenz[j]
aceanthrylene (3- methyl-cholanthrene) between 1934 and 1953, 410 studies involving B[a]P
between 1932 and 1953, 275 studies involving DB[a,h]A between 1930 and 1953, and 91
studies involving DMB[a]A between 1938 and 1953. One of the most potent PAHs in
mouse-skin-tumor induction is 1,2-dihydro-3- methylbenz[j]aceanthrylene
(3-methylcholanthrene) which theoretically could be formed pyrogenetically from sterols such as
cholesterol {Ia}. In addition to the trace level of cholesterol present in tobacco, tobacco usually
contains substantial levels of several phytosterols [campesterol {Ib}, β-sitosterol {Ic},
stigmasterol {Id}, ergosterol {Ie}] structurally similar to cholesterol. These phytosterols differ from
cholesterol {Ia} in the structure of the long side chain. Stigmasterol {Id} is structurally similar to
β-sitosterol {Ic} except for a double bond at the C22 carbon. The legend to Figure 25.1 indicates
the differences among cholesterol, campesterol, β-sitosterol, ergosterol, and stigmasterol.
These sterols, present in tobacco in both the free and bound form (as glycosides, esters), are
transferred intact to MSS. These sterols constitute about 0.2% of the tobacco weight. Table
25.7, adapted from Grunwald et al. (1971), illustrates the relative proportions of these sterols in
tobacco. These data indicate that cholesterol and cholesteryl derivatives are the least plentiful of
the free and bound sterols in tobacco. These levels are similar to those of the standard and
reference cigarettes in the National Cancer Institute (NCI) “Less Hazardous” Program (1329,
1330, 1332 1333, 2683). As shown in Figure 25.1, pyrolysis of cholesterol {Ia} yields chrysene
{III} and a Diels hydrocarbon {IV}, a methylcyclopentaphenanthrene. Both PAHs have also been
isolated from pyrolysates of the major tobacco phytosterols. While cholesterol {Ia} and the
tobacco phytosterols [campesterol {Ib}, β-sitosterol {Ic}, stigmasterol {Id}] have not been shown
to generate 3- methylcholanthrene {II} on pyrolysis, cholesterol {Ia} and its esters with
long-chained acids do generate the mouse-skin tumorigens 4-cholesten-3-one {Va} and
3,5-cholestadiene {VIa} (1171). Veldstra (4042a) reported that the pyrolysis of cholesteryl oleate
also yielded 3,5-cholestadiene {VIa}. Cholesteryl oleate was probably a component of the
mixture of steryl esters described in fluecured tobacco by Rowland and Latimer (3358) and in
tobacco smoke by Rodgman et al. (3296). The steryl esters included sterols esterified with a
series of saturated (palmitic, stearic, etc.) and unsaturated (oleic, linoleic, etc.) acids. In the late
1950s, Rodgman proposed that on thermal degradation during the smoking process,
campesterol, stigmasterol, and β-sitosterol and their esters might generate the ketones {Vb, Vc,
Vd} and dienes {VIb, VIc, VId} analogous to 4-cholesten-3-one {Va} and 5-cholestadiene {VIa},
and they might also be mouse- skin tumorigens like their cholesterol counterparts. Several
PAHs other than chrysene and Diels hydrocarbon (Figure 25.1) were subsequently identified in
sterol pyrolysates. In 1959, Wynder et al. (4355, 4356) reported that PAHs were generated at
both temperatures when tobacco sterols were pyrolyzed in air at 720°C and 850°C. At these
temperatures, the pyrolysates constituted 28% and 22%, respectively, of the phytosterols
pyrolyzed; B[a]P constituted 0.1% and 1.0%, respectively, of the pyrolysates. These results with
phytosterols pyrolyzed at two different temperatures are similar to those reported for the
pyrolyses of aliphatic tobacco hydrocarbons (2255–2258).

In 1962, Van Duuren (4022) described the identification of pyrene and B[a]P in a stigmasterol
pyrolysate. Badger et al. (142) in their pyrolysis study of tobacco phytosterols reported the
identification of some 30 PAHs, all previously reported as CSC components. They also noted
the accentuated production of chrysene vs. its generation by pyrolysis of aliphatic tobacco
hydrocarbons. Chrysene, reported to be tumorigenic to mouse skin, is a sterol pyrolysis product
characteristically generated at a high level compared to that for other PAHs in the pyrolysate.
The four-ring arrangement in chrysene is similar to that of the sterol rings. The International
Agency for Research on Cancer (IARC) eventually removed chrysene from the tumorigen list.
From a precursor “spiking” experiment involving addition to tobacco of
aliphatic tobacco hydrocarbons, a phytosterol (β-sitosterol), or solanesol, it was noted that the
increase in the chrysene yield in the CSC was much more pronounced with phytosterol-treated
tobacco than with aliphatic hydrocarbon- or solanesol-treated tobacco (3251, 3269, 3291).
Doubling the levels of solanesol, aliphatic tobacco hydrocarbons, and phytosterols by addition of
each to a control tobacco blend resulted in increases in the B[a]P yields in the mainstream
CSCs of 13%, 13%, and 16%, respectively. However, the chrysene yields were increased by
16%, 28%, and 183%, respectively. Tripling the addition levels increased the B[a]P levels in the
mainstream CSCs by 18%, 20%, and 28%, respectively, and the chrysene levels by 22%, 50%,
and 239%, respectively. In their study of the petroleum-ether-extractable material (8% of
tobacco weight) from tobacco which was chromatographically separated into eight fractions (see
Table 25.8), Severson et al. (3616) identified PAHs in the pyrolysates from Fractions F-2 and
F-3 (containing phytosterol derivatives) and from Fractions F-5 and F-6 (containing unbound
phytosterols). Their PAH data for these four pyrolysates from phytosterol-rich tobacco fractions
showed high yields of chrysene vs. those in the pyrolysates from the essentially phytosterol-free
fractions (F-1, F-2, F-7, and F-8).

Although their study dealt with pyrolysis of tobacco phytosterols, Schmeltz et al. (3511) did
determine the percent conversion of phytosterols (and other components) to MSS PAHs by use
of radiolabeled phytosterols generated by growing tobacco in an atmosphere containing
radiolabeled CO2, isolating radiolabeled tobacco components, and adding them individually to
cigarettes which were then smoked and the MSS analyzed. Their data are summarized in Table
25.9. Their 1978 finding with radiolabel techniques (<1% conversion to PAHs) for the tobacco
phytosterols is comparable to the 1958 data of Rodgman and COOK (3269, 3291) who, using
classical chemical techniques in a “spiking” experiment, reported the conversion of β-sitosterol
to PAHs to be about 0.6%, As noted by Wynder and Hoffmann (4319, 4332), the first group of
tobacco components studied by pyrolysis was the “tobacco paraffins.” Subsequently, these
were shown to consist of a mixture of n- (normal), iso- (2-methyl-), and anteiso- (3- methyl-)
saturated hydrocarbons CnH2n+2, the bulk of which comprised hydrocarbons ranging from 10
or 12 to more than 40 carbon atoms. These hydrocarbons were also extractable from tobacco
by pentane, hexane, or petroleum ether. Pyrolysis studies in air or an inert atmosphere (N2, He)
with either the tobacco-derived saturated aliphatic hydrocarbon fraction or individual
components of the fraction [see Lam (2255, 2256, 25A39), Wynder et al. (4356)] or individual
hydrocarbons [see Lam et al. (2260) for n- pentacosane, Haefele and Giles (1480) for
n-hentriacontane, Badger and Novotny (151) for n-decane, Badger et al. (142) and Lam (2256)
for n-dotriacontane (dicetyl)] indicated that these tobacco components yielded pyrolysates
reported to be tumorigenic in mouse-skin-painting bioassays and to contain many PAHs,
several of which were tumorigenic in long-term mouse-skin-painting bioassays. The PAHs
identified in the various pyrolysates ranged in complexity from bicyclic (naphthalenes), tricyclic
(acenaphthenes, anthracenes, phenanthrenes), tetracyclic (pyrenes, fluoranthenes, chrysenes,
benzanthracenes), pentacyclic (perylenes, benzopyrenes, dibenzanthracenes,
benzofluoranthenes), hexacyclic (dibenzochrysenes), and heptacyclic (coronene). Eventually,
all the PAHs identified in the various pyrolysates were identified in mainstream CSC. In every
case where the pyrolysis temperature was lowered, the yields of the PAHs in the pyrolysate also
decreased (see Table 25.6).

From data they generated, Rayburn and Wartman (3091) and Rayburn et al. (3092) challenged
the concept that the saturated aliphatic hydrocarbons in tobacco were precursors of the PAHs in
mainstream CSC. Wynder and Hoffmann (4319, 4332), in turn, criticized the experimental
procedures which provided the data upon which Rayburn et al. based their argument:
The experimental findings [of Rayburn et al.] are partially based on total polycyclic hydrocarbons
of similar ultraviolet spectra and not on analytical data. The report did not mention counting
techniques for C14- labeled paraffins nor their quenching effects. These, as well as other
factors, appear to weaken considerable the challenge of a concept based on extensive
experimental data.

In 1979, Severson et al. (3616), in their study of the PEE (8% of tobacco weight)
chromatographically separated into eight fractions (see Table 25.8), reported the identification of
PAHs in the pyrolysate from Fraction F-1, the fraction containing the saturated aliphatic
hydrocarbons extracted from the tobacco. As recently as 1985, Lam et al. (2260) identified
numerous PAHs in the pyrolysate from n-pentacosane, a known component of the saturated
aliphatic hydrocarbon fraction present in tobaccos. In 1958, Rodgman and Cook (3269, 3291)
added tobaccoderived saturated aliphatic hydrocarbons to a tobacco blend in a “spiking”
experiment and determined the effect of the addition on the PAH levels in mainstream CSC.
They reported the added saturated hydrocarbons increased the yield of PAHs in cigarette MSS
and thus were precursors of the smoke PAHs. The structure of the unsaturated C45
polyisoprenoid alcohol, solanesol, was established in 1956 by Rowland et al. (3359). Despite
the fact that solanesol was one of the major individual components of the extractable waxes
from tobacco, its pyrolysis was not reported until 1962. While Lam (2255) favored the saturated
hydrocarbons as the major precursors in tobacco of PAHs in smoke, Wynder (4294) considered
both the saturated hydrocarbons and the phytosterols to be the major precursors. Wright (4282)
proposed that the phytosterols and other terpenoids such as solanesol were the major
precursors in tobacco of PAHs in smoke. In spite of their differences of opinion on the relative
importance of these tobacco components in their contribution to smoke PAHs, they collaborated
on several studies in the late 1950s (4355, 4356). Subsequently, the saturated hydrocarbons,
the phytosterols, and other terpenoids such as solanesol were shown to be important in the
formation of PAHs in tobacco smoke (3251, 3269, 3291, 3616). In the early 1960s, Grossman et
al. (1431, 1432) examined the pyrolysate from solanesol and reported the identification of
monocyclic hydrocarbons (benzene and cyclopentene derivatives) (1431) and bicyclic aromatic
hydrocarbons (naphthalenes) (1432). No tricyclic PAHs were reported. In 1963, Gil-Av and
Shabtai (1286) postulated that solanesol in tobacco was a source of tobacco smoke PAHs and
proposed a mechanism for their generation from solanesol: Solanesol and other similarly
configured terpenoid compounds, eg, neophytadiene, squalene, duvane derivatives,
depolymerized during the smoking (or pyrolytic) process to produce isoprene which, in turn,
reacted with itself and subsequent reaction products to generate a tumorigenic “tar” such as that
described in the mid1920s by Kennaway (2073–2075). This “tar” derived from solanesol via
isoprene would contain the requisite tumorigenic PAHs such as B[a]P. Although Gil-Av and
Shabtai (1286) demonstrated the presence of B[a]P in an isoprene pyrolysate, they did not
study the pyrolysis of solanesol. Severson et al. (3616) described the pyrogenesis of PAHs from
solanesol in their study of the petroleum ether tobacco extractables (8% of tobacco weight)
which they chromatographically separated into eight fractions (see Table 25.8). Fraction F-4
was primarily solanesol. Fraction F-3 contained solanesyl esters. Severson et al. (3616)
summarized the contribution of solanesol to PAHs in its pyrolysate (and in CSC):

The carotenoids and solanesol are most like responsible for the high levels of the
multialkylated PAH found in the [petroleum ether]-extract pyrolyzate and, by analogy, in
CSC. Because of its relative abundance in leaf, solanesol may contribute as much as
40% of the benzopyrenes produced on pyrolysis of the [petroleum ether] extract of
tobacco.
As noted previously, 1958 precursor experiments (3251, 3269, 3291), in which solanesol was
added at several levels in a “spiking” experiment and the effect of this addition on the levels of
total and individual PAHs in cigarette MSS was determined, demonstrated that solanesol in
tobacco was indeed a significant precursor of PAHs in cigarette MSS. Phytol, a terpenoid
alcohol, is a known component of tobacco leaf. It and its structurally similar dehydration product,
neophytadiene, probably occur in tobacco leaf through the degradation of chlorophyll whose
structure includes phytol (3345). Neophytadiene (3247), phytol (3285), and phytyl esters (3287)
are present in tobacco smoke (see Table 25.5). Schmeltz et al. (3511), in their 1978 radiolabel
study, determined the contribution of neophytadiene to PAHs in cigarette MSS. They estimated
that about 0.1% of the tobacco neophytadiene is converted during the smoking process to PAHs
in the MSS (see Table 25.9). The pyrolysis products of neophytadiene and phytol were
examined in 1985 by Lam et al. (2260). Numerous PAHs were identified in the pyrolysates of
both compounds. However, as a result of their study of the pyrolysis of phytol and a phytol
derivative, Moldoveanu et al. (5539) considered in 2010 that phytol was not a significant
contributor to PAHs in cigarette smoke, a view that disagreed with the findings of Lam (2260)
and Schlotzhauer and Schmeltx (3466) and the earlier views and findings of Wright (4282),
Rodgman (3251), Rodgman and Cook (3269, 3291), Schmeltz et al. (3511), and Severson et al.
(3616) on the contribution of polyisoprenoid components in tobacco to the levels of PAHs in
cigarette smoke. Normal long-chained aliphatic alcohols, known minor components of tobacco
(614, 615, 25A18, 25A80) and tobacco smoke (812), may not play a significant role as PAH
precursors. In fact, Carruthers and Johnstone (614, 615), who identified 1-docosanol in tobacco,
found little of its dehydration product, 1-docosene, in tobacco smoke. They postulated that
long-chained primary alcohols such as 1- docosanol were little affected by pyrolysis. Severson
et al. (3616) cataloged the major components in eight chromatographic fractions of the PEE
from tobacco (see Table 25.8). Each fraction was subjected to pyrolysis. Surprisingly, Severson
et al. did not list long-chained primary alcohols in any of the eight chromatographic fractions!

25.3.3 Structural compoNeNtS of tobacco (celluloSe, ligNiN,

pectiNS, etc.)

Since cellulose, pectins, starch, lignin, and proteins—the socalled structural components
of tobacco—are organic compounds, ie, contain carbon, with one or more of the carbons linked
to hydrogen,* they will generate PAHs during high-temperature pyrolysis much in the same
manner as the compounds studied in the mid-1920s to early 1930s by Kennaway (2073– 2076).
In his studies, Kennaway demonstrated that pyrolysis of various organic compounds—from
simple ones such as acetylene or isoprene to more complex ones—would generate pyrolysates
tumorigenic to mouse skin. Subsequently, these and similar organic compound-derived
pyrolysates were shown to contain a variety of PAHs (1286, 2264b), some of which were potent
mouse-skin tumorigens. Obviously, PAHs should be generated from the structural components
of tobacco during the reactions occurring when tobacco is smoked in a cigarette. Interest in the
major precursors in tobacco of the PAHs in MSS eventually led to the conclusion that the major
precursors in tobacco of cigarette MSS PAHs in cigarette MSS were the organic solvent-
extractable, high-molecular-weight tobacco components such as the saturated aliphatic and
unsaturated aliphatic hydrocarbons, the phytosterols, terpenoid alcohols such as solanesol
(3251, 3269, 3291, 3616, 4332). During the search for the major PAH precursors in tobacco, all
of the previously mentioned organic solventsoluble tobacco components [cf. Table 25.5] that
were examined by pyrolysis were shown to yield PAHs. In 1957, Gilbert and Lindsey (1289)
reported their results on the amounts of various PAHs in the pyrolysates (650°C, N2) from the
major structural components from flue-cured tobacco. These included cellulose, pectins, starch,
and lignin; the simple sugars, sucrose, glucose, and fructose; and the acids, malic
acid, citric acid, and oxalic. Their data, modified to indicate nanograms of individual PAHs
generated per milligram of tobacco component pyrolyzed,† are summarized in Table 25.10.
Numerous pyrolysis experiments were conducted from the mid-1950s to the mid-1980s on
tobacco components and tobacco fractions and residues obtained by solvent extraction of
tobacco. However, meaningful comparison of the results has been difficult because of the lack
of uniformity in the pyrolysis conditions employed in the studies. Even when some similarity
existed between experimental conditions used in two separate experiments, precise comparison
was confounded by the fact that different tobaccos or blends were used in the experiments, eg,
Gilbert and Lindsey (1289) examined the PAH yields in the pyrolysates (650°C, N2) from the
structural components of a flue-cured tobacco grown a year or two prior to their 1957 study;
Severson et al. (3616) examined the pyrolysates (produced at a variety of temperatures
including 650°C, N2) from a flue-cured tobacco probably grown a few years before their 1979
publication and from a tobacco blend. In their detailed study, Severson et al. (3616) examined
the following pyrolysates: a flue- cured tobacco; its PEE (8% of the tobacco weight); the tobacco
residue after petroleum ether extraction; and eight chromatographic fractions (Fractions F-1
through F-8) derived from the PEE. Unfortunately, Severson et al. conducted several key
pyrolysis experiments at 700°C only. It should also be noted that tobaccos, such as the
flue-cured tobaccos, used by Gilbert and Lindsey and by Severson et al. would not be identical
because of the differences in agronomic conditions and practices for the tobaccos grown in the
mid-1950s vs. the mid-1970s. Table 25.11 summarizes pyrolysis data from Lam (2257), Gilbert
and Lindsey (1289), and Severson et al. (3616) with particular emphasis on the somewhat
similar experimental conditions (pyrolysis temperature, atmosphere) and on the yields of B[a]P
and B[e]P from the pyrolysis of different tobaccos, blend, components, and/or fractions. The
data in Table 25.11, plus additional data in the publications cited, indicated that the structural
components as well as other components and fractions (organic solvent-soluble or -insoluble)
from tobacco yielded a variety of PAHs on pyrolysis, but the structural components—the
biopolymers—such as cellulose, pectins, starch, lignin, on a per unit weight pyrolyzed basis
generated much lower yields of PAHs than did the organic solvent-soluble components and/or
fractions. Even in the cases where tobacco components are not only soluble in organic solvents
but also are relatively highly oxygenated, they show a low propensity to generate PAHs on
pyrolysis. This fact was demonstrated by Severson et al. (3616) in their study of the pyrolysates
from the eight chromatographic fractions (Fractions F-1 through F-8) from the PEE from
fluecured tobacco. They noted:

Extraction fractions F-1 and F-8 yielded relatively low yields of PAH on pyrolysis, the
former because of its thermally stable hydrocarbon content, and the latter because of its
polar oxygenated constituent content. Such oxygenated compounds, having a relatively
low carbon content yield low amounts of the alkyl residues essential for PAH formation
[see Badger et al. (148), Schmeltz and Hoffmann (3489)]...

The Fraction F-8 components which Severson et al. in their pyrolysis studies demonstrated to
have a low propensity to yield PAHs on pyrolysis are similar both structurally and property-wise
(molecular weight, volatility) to some of the compounds used in “top dressing” formulations for
tobacco smoking products [see tabulations in Doull et al. (1053), Leffingwell et al. (2341)].
Presumably, “top dressing” components applied to tobacco products on pyrolysis would behave
similarly during pyrolysis to the Fraction F-8 oxygenated tobacco components described by
Severson et al. [see pp. 284–285 in (3616)]. Even though the structural components of tobacco
on pyrolysis did yield PAHs, albeit at a much lower level than other classes of tobacco
components, their contribution to tobacco smoke composition became important from
another point of view: When only a small portion (about 2%–3%) of the biological response
observed in mice (or other rodents) skin painted with CSC could be explained by its content of
the PAHs reported to be tumorigenic to mouse skin [see pp. 14–51/52 in (4005), Wynder et al.
(4303)], additional explanations for the observed biological response were sought. The concepts
of promotion and cocarcinogenesis were introduced into the theory of CSC tumorigenicity in an
attempt to explain the observed biological response in the mouse-skinpainting studies. In the
1950s, Boutwell et al. (414) and Boutwell and Bosch (414) reported that low-molecular-weight
phenols such as phenol itself and the cresols, nontumorigenic per se in skin-painting
experiments, enhanced the tumorigenicity in mouse-skin-painting studies of PAHs reported to
be tumorigens. In 1959, Roe et al. (3314) reported the promoting effect of a phenolic fraction
from cigarette MSS.

Two years later, Wynder and Hoffmann (4313) examined phenol as a promoter of several PAHs
(B[a]P, DMB[a]A) and concluded:

Promoting substances present in tobacco smoke can increase and accelerate the tumor
yield of carcinogenic polynuclear hydrocarbons that by themselves are not present in
sufficient concentration to yield any tumors or yield them only after a prolonged latent
period.

The low-molecular-weight phenols are extremely low-level components of tobacco but are
present in cigarette MSS at levels many times those in tobacco. This led to the search in the
late 1950s to early 1960s for precursors in tobacco of the alleged biologically active phenols in
tobacco smoke. Studies by Rodgman and Cook (3277), Rodgman and Mims (3305), and
Rodgman (3251) on the effect of tobacco components (lignin, pectin) added to a cigarette
tobacco blend on low-molecular-weight phenol levels in MSS and similar studies [Spears et al.
(3767), Bell et al. (248)] on the effect of tobacco carbohydrates (glucose, sucrose, starch,
cellulose, or pectin) added to cigarette tobacco filler on phenol levels in MSS demonstrated that
these tobacco components were major precursors of the simple phenols in cigarette MSS. From
1962 through 1971, Newell and Best conducted studies with radiolabeled components isolated
from tobaccos grown in a radiolabeled CO2 atmosphere. Among these radiolabeled
components studied were the cell wall components or biopolymers [pectin (2764), starch (2764),
α-cellulose (2764)] of the tobacco. Each component was added individually to cigarette tobacco,
and their contributions to various classes of MSS components, particularly the PAHs and the
phenols, were determined. Table 25.12 summarizes the Newell–Best findings on the percent
conversion during smoking of these three structural components to specific PAHs and phenols.
Examination of these data indicates that the percent conversion of these structural components
to phenol ranges from one (0.0013%) to about four (0.0036%) one thousandths of a percent,
whereas the conversion of these three components to B[a]P ranges from about 1 (0.0000014%)
to about 14 (0.0000136%) one millionth of a percent. Such data may be used to estimate the
conversion to PAHs and phenols of a flavorant (structurally similar to but of lower molecular
weight and higher volatility than these biopolymers) added to the tobacco blend. Numerous
pyrolysis studies were conducted on these precursors of phenols. In each case, the generation
of simple phenols [phenol, cresols, numerous xylenols] was observed. Kato et al. (2043)
reported the pyrolysis of tobacco lignin yielded phenol, cresols, xylenols, and guaiacol—all
known components of cigarette MSS. Examination of the structure of lignin reveals why it would
readily yield these phenols as well as other substituted phenols such as vanillin [Ball (176a)].
From the mid-1960s to the early 1980s, the USDA tobacco research group—initially at
Philadelphia, PA, subsequently at Athens, GA—described the pyrolysis of various tobacco
components and the yield of phenols in the pyrolysates. Tobacco components examined for
their propensity to generate the simple phenols during pyrolysis included the following:
• Cellulose, pectin, lignin, and the so-called tobacco pigment
(3468).
• The major tobacco polycarboxylic acids—malic, citric, and fumaric plus the sodium salts of
citric and lactic acids (3486). In the late 1950s, a mixture of sodium and potassium
citrates was used as an additive on cigarette paper to control its combustion
properties.*
• Tobacco and tobacco extracts [Kennedy and Riehl (25A35), Schlotzhauer et al.
(3456), Severson et al. (3616)].
• Cellulose, glucose, and fructose [Higman et al. (1647)]. The latter two sugars† are used
in casing materials applied to cigarettes during manufacture [Leffingwell et al.
(2341)].

With regard to the generation of phenols by the tobacco acids, Schmeltz et al.
(3486) noted:
 The data show that citric, malic and related acids give rise to phenols on pyrolysis. The
yields, however, are lower than those from other phenol-forming materials present in
tobacco leaf.

Despite the repeated assertions of the promoting potency for PAHs of the CSC phenolic fraction
[Roe et al. (3314)] and the simple phenols, particularly phenol itself [Wynder and Hoffmann
(4313, 4332)], contradictory evidence was reported. In 1962, Bock and Moore (25A11)
challenged the concept that the weakly acidic portion of CSC was a tumor promoter. In fact,
Wynder and Hoffmann (4319), strong proponents in 1964 of the promoting effect of the phenols
in smoke, wrote:

Definite tumor-promoting activity for a variety of phenols may be regarded as

established​.

However, this statement was omitted from their 1967 book in which Wynder and Hoffmann [see
p. 626 in (4332)] wrote:

Phenol and some of its derivatives have been shown to possess tumor-promoting
activity...However, a reduction of phenols in tobacco smoke condensate has not led to a
concomitant reduction of tumorigenicity in the corresponding 'tars'.

In 1971, Van Duuren et al. (4035), in a discussion of tumor promoters and the complexity of
CSC and its potential role in carcinogenesis, reported that “Phenol, which is a weak
tumor-promoting agent, is indeed an inhibitor of tumorigenesis when applied simultaneously
with benzo[a]pyrene.” Two years later, Van Duuren et al. (4029) concluded from their
cocarcinogenesis research that “Phenol has been regarded as an important 'tumor promoter' in
[cigarette smoke condensate]...[but our] work indicates that it is inactive in cocarcinogenesis
and, indeed, has a slight inhibitory effect on benzo[a]pyrene carcinogenesis.” Chortyk and
Schlotzhauer (722) reviewed the studies reported during the preceding two decades on
pyrolysis of tobacco components and the relationship of the pyrolysis results to the pyrogenesis
of tobacco smoke components. In 1979, Martin et al. (2468a) not only reported the results of
their own research on the generation of a variety of phenols during the pyrolysis of lignin derived
from several sources but also reviewed the results of their own and earlier studies by other
investigators on lignin pyrolysis. From their 1981 and 1982 studies on pyrolysis, Schlotzhauer
and Chortyk (3453) and Schlotzhauer et al. (3452) reported the pyrogenesis of phenols not only
from cellulose and lignin but also from chlorogenic acid and other polyphenols. Cellulose was
defined as a major precursor of cresols and xylenols in smoke; lignin as a major precursor of
guaiacol, eugenol, and catechol in smoke; the polyphenols, eg, chlorogenic acid, as major
precursors of the catechols in smoke.

In 1975, Schlotzhauer and Chortyk (3452), emphasizing the toxicants in tobacco smoke,
reported that the yields of PAHs and phenols in the pyrolysate from reconstituted tobacco sheet
(RTS) were significantly
lower than those from flue-cured tobacco leaf when generated under the same experimental
conditions. Extrapolating their pyrolysis results to the formation of specific components of
tobacco smoke, Schlotzhauer and Chortyk noted that “...the continued use of reconstituted
tobacco sheet in tobacco products appears warranted.” Comparison of data obtained under
different pyrolysis conditions with cellulose, glucose, and fructose with data generated under
actual smoking conditions reveals the problem of attempting such a comparison. In Table 25.13
are summarized data from Gilbert and Lindsey (1289), Higman et al. (1647), and Newell and
Best (2764) on the conversion (ppm or μg/g) of such tobacco components to B[a]P during
pyrolysis under two different conditions (650°C and 840°C, N2) and when smoked in a cigarette
under actual smoking conditions (35 mL puff, 2 s puff duration, 1 puff/min). The degree of
conversion of tobacco components, such as cellulose, to B[a]P under different pyrolysis
conditions parallels the degree of conversion of other tobacco components such as the
saturated aliphatic hydrocarbons to B[a]P under different pyrolysis conditions, eg, Lam (2257)
and his findings summarized in Table 25.11. The conversion of cellulose to B[a]P increases
several hundredfold as the pyrolysis temperature increased nearly 200°C (from 650°C to
840°C). Under actual smoking conditions, the cellulose-to-B[a]P conversion was less than 30%*
of the conversion at 650°C. Similarly, under actual smoking conditions, the conversion of
cellulose to B[a]P was only 0.073% of the conversion at 840°C. Thus, these data from several
sources indicate that the fate of a tobacco component on pyrolysis is not equivalent to its fate
under actual smoking conditions [cf. the conclusion of Schmeltz et al. (3512) on the fate of
nicotine on pyrolysis vs. its fate during actual smoking]. As noted in reviews of the thermal
degradation products from tobacco carbohydrates (cellulose, pectins, starch, sugars) by
Roberts et al. (3225) and by Schumacher (3551), pyrolysis of the carbohydrate components of
tobacco results in generation of several classes of compounds other than PAHs and phenols.
Among these were aldehydes and ketones, considered significant in smoking–respiratory tract
issues. It was asserted during the 1960s that aldehydes and ketones, shown to be ciliastatic in
vitro to ciliated tissue, were important because of their significant impairment (by extrapolation)
of the action of human respiratory tract cilia. Such impairment was considered part of the
mechanism of lung cancer causation by cigarette smoke. However, their importance diminished
after the reported findings of Dalhamn et al. (892) that these cigarette smoke-derived,
water-soluble in vitro ciliastats were removed in large part by the “scrubbing action” of the fluids
coating the surfaces of the oral cavity and laryngeal area, a phenomenon demonstrated several
years earlier by Rodgman et al. (3306). Early studies (1955–1959) on carbonyl components
included those of Fredrickson (1228) who examined the volatile MSS components from
all-cellulose cigarettes. Many were identified as aldehydes and ketones (1238, 1239). In 1959,
Laurene et al. (2310) reported the unequivocal identification of acrolein in cigarette smoke, and
cellulose in tobacco was a major precursor of it in smoke. Grob (1413) subsequently
demonstrated in 1962 that, during smoking, cellulose generated high levels not only of acrolein
but also the ketone 3-buten-2-one. In 1966, Latimer (25A40) reported several aldehydes
(acetaldehyde, acrolein) and ketones (acetone, 2-butanone) in the pyrolysate from
tobacco-derived starch. At Philip Morris R&D, Gager et al. (1264, 1265) noted from their study
with radiolabeled sugars added to cigarette tobacco that acetaldehyde and acetone were
formed during the smoking process in the highest yields from added sugars but their levels were
reduced because of the levels generated from other major tobacco components such as
cellulose, pectins, and starch. In their 1976 literature review of pyrolysis products from
carbohydrates, Roberts et al. (3225) noted that, of the more than 140 compounds identified in
the pyrolysates from glucose, fructose, sucrose, cellulose, and starch, 21 were aldehydes, 30
were ketones. Of these 51 pyrolysis products identified at that time, 40 had been identified as
cigarette MSS components. In 1977, Ohnishi and Kato (2850) described the
identification of several carbonyl compounds in the pyrolysates from the tobacco cell wall
polysaccharides cellulose, hemicellulose, and pectins. They noted that these biopolymeric
polysaccharides constituted 30%–50% of the dry weight of the tobacco they were studying.
Sakuma et al. pyrolyzed tobaccoderived cellulose (3401), chlorogenic acid, and rutin (3400) and
reported various aldehydes and ketones plus numerous phenols in the pyrolysates. Many of the
pyrolysate components identified have also been identified in cigarette MSS.

25.3.4
ACIDS

The research findings of Gilbert and Lindsey (1289) on the generation of a variety of PAHs
during the pyrolysis of major components of tobacco, including several polycarboxylic acids
(oxalic, malic, and citric acids), were discussed previously (see Table 25.10). These acids may
constitute from 3% to 12% of dry tobacco weight (1289, 1329, 1330, 1332, 1333). At RJRT
R&D, the fate of these acids during smoking in a cigarette was determined by Newell and Best
in studies with radiolabeled acids added individually to cigarette tobacco (2763). In 1967,
Schmeltz et al. (3486), in their attempt to define precursors in tobacco of several classes of
compounds in cigarette smoke, studied the nature and levels of phenols generated in the
pyrolysates of malic, citric, aconitic, and fumaric acids or their sodium salts. Thus, pyrolysis of
tobacco leaf acids yielded phenols (3486) and PAHs (1289). Although direct comparison is
somewhat difficult because of the 50°C pyrolysis temperature difference in the Gilbert– Lindsey
vs. Schmeltz et al. studies, the PAHs appeared to be generated in much lesser amounts per
gram of tobacco leaf acid pyrolyzed than were the phenols. Table 25.14 summarizes data
obtained from the studies. Pyrolysate products obtained from several short-chained aliphatic
acids or their sodium salts were examined. Schmeltz and Schlotzhauer (3498) pyrolyzed sodium
acetate at 500°C and 800°C. The 800°C pyrolysate was much more complex than that
produced at 500°C. This was true for the numbers of pyrolysate components generated and
those with aromatic structures. Among the latter were several alkylbenzenes and phenols.
Rudenko and Konsinska (25A61) reported similar findings from their pyrolysis of propionic acid,
present in tobacco in bound form. At the USDA, Geisinger et al. (1279) demonstrated that
pyrolysis of malic and lactic acids at temperatures from 500°C to 900°C (in 100°C increments)
yielded both aromatic hydrocarbons and phenols. With malic acid, the aromatic hydrocarbon
complexity increased (increased methylation) as the temperature increased. Bicyclic indene was
the only PAH detected in the 500°C and 600°C pyrolysates. Indene and naphthalene were
detected in the 700°C pyrolysate. Tricyclic acenaphthylene, anthracene, phenanthrene, and
fluorene were detected in the 800°C pyrolysate, tetracyclic pyrene, and chrysene in the 900°C
pyrolysate. Pyrolytic products from several aromatic acids present either free or bound in
tobacco were investigated, eg, Zane and Wender (4403) demonstrated in 1963 that pyrolysis of
rutin, quercetin, and chlorogenic acid (tobacco polyphenols with bound caffeic acid) yielded
catechol as the major product, alkylcatechols, resorcinol plus several furancarboxaldehydes.
Similar results were reported by the USDA group (3462) at Athens, GA. In 1969, Jones and
Schmeltz reported catechol as the major pyrolysis product (32%) from free caffeic acid (1981)
and stilbene as the major pyrolysis product from trans-cinnamic acid (1983). trans-Cinnamic
acid pyrolysate also contained low yields of several bicyclic and tricyclic PAHs. The results of
these and similar pyrolysis studies with tobacco acids were reviewed by Chortyk and
Schlotzhauer (722). Indirect evidence that major leaf acids such as malic, citric, and oxalic acids
in tobacco contributed low PAH levels to pyrolysates from tobacco fractions was provided by
Severson et al. (3616). When tobacco was extracted with hexane or petroleum ether, the bulk of
these acids did not appear in the extract but remained in the insoluble tobacco residue.
Severson et al. reported that the pyrolysate from the extractables (8% of tobacco weight)
contained more
than twice the amount of total PAHs than did the pyrolysate from residual tobacco (92% of
tobacco weight). In the same study, Severson et al. (3616) examined the levels of a various
PAHs (bicyclic to pentacyclic) in the pyrolysates of eight chromatographic fractions from the
PEE. Several fractions consisted primarily of free fatty acid mixtures such as myristic, palmitic,
stearic, oleic, and linoleic acids (Fractions F-5 and F-6) and esters of longchained saturated and
unsaturated alcohols with these acids (Fractions F-7 and F-8, see Table 25.8). As described
previously, Schmeltz et al. (3511) demonstrated that less than 1% of radiolabeled palmitic acid,
isolated from tobacco grown in a radiolabeled CO2 atmosphere and added to cigarette tobacco
filler, was converted to PAHs during the smoking process. Pyrolysis products from esters such
as ethyl acetate and isobutyl acetate, both possible flavorants for tobacco smoking products,
have been reported [Leffingwell et al. (2341), Miyagawa (2563)]. The principal pyrolysis
products from both acetates were CO, CO2, methane, acetic acid, and acetone. Isobutyl
acetate yielded isobutylene as a major product. Products from pyrolysis of the esters formed
from long-chained fatty acids* and glycerol (triglycerides) were described by Higman et al.
(1646) (tripalmitin, tristearin) and Kitamura (2111a) (trilaurin, tripalmitin). In the early 1970s,
Halaby and Fagerson (25A28) pyrolyzed palmitic, oleic, and linoleic acids plus their
triglycerides. Numerous PAHs were identified in the pyrolysates. They reported that B[a]P was
generated from each acid and from each triglyceride at about 100 ppm of the compound
pyrolyzed. In their studies on precursors in tobacco of PAHs in tobacco smoke, Rodgman and
Cook (3269) demonstrated that addition of 0.4% (4.0 mg/g of tobacco) of trimyristin to tobacco
produced, under actual smoking conditions, a 6% increase in total PAHs in the MSS. The
changes in individual PAH yields are shown in Table 25.15. These changes are also expressed
in Table 25.15 in terms of the conversion (ng/mg or ppm) of the added trimyristin to individual
PAHs. The changes observed in the levels of individual PAHs are well within the experimental
error for PAH analyses in the late 1950s.

25.3.5 PROTEINS AND AMINO

ACIDS
Amino acids, both as free acids and as acids bound within protein molecules, are present in all
of the tobacco types (flue-cured, burley, Oriental, Maryland). The diversity and levels of amino
acids in various tobaccos have been presented by Gori (1329, 1330) and Tso and Chaplin
(3975). The amino acids occurring free and/or bound in tobaccos include alanine,
α-aminobutyric acid, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, ornithine, phenylalanine (Phe), proline, serine, threonine, tryptophan
(TRY), tyrosine, and valine. The presence in cigarette MSS of numerous free amino acids and
amino-acid-derived compounds was demonstrated in the mid-1950s. This occurred soon after
the publication of the results of several cigarette-smoke-related epidemiological and biological
studies led to a massive escalation in tobacco smoke composition studies; eg, Buyske et al.
(562) identified glutamic acid and its derivative glutamine (glutamic acid 5-amide) in tobacco
smoke. Other amino acids identified in tobacco smoke [cf. Ishiguro and Sugawara (1884)]
include alanine, aspartic acid (and asparagine), cysteine, glycine, leucine, ornithine, Phe,
proline, serine, threonine, and valine. In the early 1960s, pyrocoll
(dipyrrolo[a,d]pyrazine-5,10dione) was identified in cigarette MSS by Mold et al. (2592) who
proposed that either free or bound proline was its precursor. During their study of the isolation
and identification of N-heterocyclic components (the indoles and carbazoles) in cigarette MSS,
Rodgman and Cook (3279) confirmed the presence of pyrocoll. Two decades earlier, Van Order
and Linwall (25A78) had demonstrated that dry distillation of TRY yielded indole and
3-methylindole (skatole), both of which were subsequently identified in tobacco smoke (3279)
and in burley tobacco by Roberts [see citation in Rodgman and Cook (3279)]. From their
pyrolysis studies (850°C, N2) with lysine, leucine, and TRY, Patterson et al. (2902) reported that
each yielded the N-heterocyclic compounds indole, quinoline,
isoquinoline, several nitriles, and PAHs ranging from bicyclic to tetracyclic (see Table 25.16).
B[a]P was found only in the leucine pyrolysate. From their own findings and from a previous
report by Jarboe and Rosene (1923a) that quinoline and isoquinoline were components of a
nicotine pyrolysate Patterson et al. suggested that the precursors in tobacco of quinoline and
isoquinoline in tobacco smoke might be nicotine and/or the amino acids. They also reported that
TRY, per mole pyrolyzed, yielded a phenol fraction weighing about five times that generated
from lysine and about 30 times that from leucine. Patterson et al. (2903) reported the effect of
temperature on the pyrolysate composition from Phe, with emphasis on PAHs yields, and the
effect of TRY or pyrrole (PYR) on the pyrolysate composition when equimolar quantities of Phe
+ TRY or Phe + PYR were pyrolyzed (see summary of results in Table 25.17). The difference
between the pyrogenesis of PAHs from Phe and equimolar quantities of Phe + TRY mixture
prompted Patterson et al. (2903) to propose amino acid addition to tobacco to control the PAH
content of the CSC:

These results suggest the possibility that aromatic hydrocarbon content of tobacco “tar”
may be affected by the amino acid composition of the tobacco and that it might be
possible to affect deliberately the amount of aromatics and bases formed by adding
suitable additives, such as amino acids, to the tobacco.
In 1971, when Patterson et al. made this suggestion, the presence in amino acid pyrolysates of
the N- heterocyclic amines and the inordinately high mutagenicity of several of them were
unknown. Higman et al. (1647) reported the generation of PAHs, phenols, pyridines, indole,
quinoline, and other aromatic bases during pyrolysis of tobacco amino acids and proteins [cf.
review on pyrogenesis of smoke components by Chortyk and Schlotzhauer (722)]. The results
reported by Higman et al. are summarized in Table 25.18. TRY was found to be the precursor in
tobacco of harman (1-methyl-9H-pyrido[3,4-b]indole) and norharman (9H-pyrido[3,4-b]indole) in
tobacco smoke, compounds originally identified in tobacco and tobacco smoke by Poindexter
and Carpenter (2972). That TRY was indeed a precursor in tobacco of the harmans in smoke
was demonstrated by addition of radiolabeled TRY to tobacco and identification of radiolabeled
harman and norharman in the MSS. More recent amino acid pyrolysis studies led to the
isolation and identification of several N-heterocyclic amines reported not only to be tumorigenic
to mouse skin but also to show inordinately high mutagenicity [Ames bioassay (S.
typhimurium)]. Initial impetus for amino acid pyrolyses was not the definition of the relationship
between tobacco precursors and smoke components but the observation that extracts of
broiled, fried, or roasted foodstuffs were highly mutagenic (Ames bioassay). These
N-heterocyclic amines, derived from amino acids and/or proteins in heated foodstuffs, were
defined as “cooked food” mutagens. These studies in the 1970s on the tumorigenicity and
mutagenicity of extracts of cooked foodstuffs are reminiscent of the studies in the 1920s by
Kennaway (2073–2076) who reported the tumorigenicity of extracts of heated foodstuffs or
pyrolysates from compounds such as cholesterol and by Roffo (25A56, 25A57, 25A58) who
reported the tumorigenicity of pyrolyzed cholesterol. Subsequently, pyrolysates from many
foodstuffs and cholesterol were shown to contain various PAHs, including B[a]P. Identification of
highly mutagenic N-heterocyclic compounds in amino acid pyrolysates was followed by their
identification not only in heated foodstuffs but also in mainstream CSC. In 1977, Sugimura et al.
(3829) reported the identification of the potent mutagens 3-amino-1-methyl-5H-pyrido[4,3-b]
indole (Trp-P-2) and 3-amino-1,4-dimethyl-5H-pyrido [4,3- b]indole (Trp-P-1) in TRY pyrolysate.
The next year, Yamamota et al. (4365a) identified two potent mutagens in glutamic acid
pyrolysate: aminodipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-2) and 2-amino-6-
methyldipyrido[1,2-a:3′,2′-d] imidazole (Glu-P-1). Table 25.19 lists several N-heterocyclic amines
which
exhibit high mutagenicity in the Ames bioassay, are amino acid pyrolysis products, and have
been identified in heated foodstuffs and CSC (3828c). On a per μg basis, B[a]P in the Ames
bioassay with S. typhimurium (TA 98 strain) shows about 200 revertants/μg. Several of the
amino-acid-derived compounds in Table 25.19 exceed the B[a]P effect (TA 98 strain) by factors
ranging from about 10 to over 2100.

Yoshida and Matsumoto (4387a) reported the identification of two α-carbolines in CSC:
2-amino- 9H-pyrido[2,3- b] indole (AαC) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC).
These and several other compounds were reported in CSC by Yamashita et al. (4367, 4368).
The quantitative levels of the possibly amino-acid-derived, mutagenic N-heterocyclics in CSC
are shown in Table 25.19. In their studies, they emphasized in particular the identification and
quantitation of 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) because of its inordinately high
mutagenicity (433,000 and 490,000 revertant/μg in the Ames bioassay, S. typhimurium strain
TA 98). Demonstration of the mutagenicity of the compounds in Table 25.19 was followed by
demonstration of their tumorigenicity in laboratory animals. Ohgaki et al. (2849a) demonstrated
the tumorigenicity of IQ in mice. Takayama et al. (3862c) and Tanaka et al. (3865c) reported its
tumorigenicity in rats. Trp-P-1 and Trp-P-2 were reported to be tumorigenic in mice by
Matsukura et al. (2491a) and in rats by Hosaka et al. (1835a) and Takayama et al. (3862d).
Ohgaki et al. (2849b) reported that Glu-P-1, Glu-P-2, AαC, and MeAαC were tumorigenic in
mice, and Takayama et al. (3862b) reported Glu-P-1 and Glu-P-2 to be tumorigenic in rats.
Hoffmann and Hecht (1727) discussed the amino-acidderived aromatic amines in cigarette
smoke as follows:

Of the known carcinogenic pyrolysis products of the amino acids, so far only 2-amino-3-
methylimidazo(4,5-f)quinoline has been detected in trace amounts of 0.26 ng in the
smoke of a Japanese filter cigarette [Yamashita et al. (4368)].

Apparently, Hoffmann and Hecht had overlooked not only the reports of the identification in CSC
of several other known “carcinogenic” pyrolysis products of amino acids, eg, AaC and MeAaC
[Yoshida and Matsumoto (4387a)] or Trp-P-1 and Trp-P-2 [Yamashita et al. (4367)], but also the
reports on their tumorigenicity in several animals species [Matsukura et al. (2491a), Hosaka et
al. (1835a), Ohgaki et al. (2849a, 2849b), Takayama et al. (3862c, 3862d), Tanaka et al.
(3865c)]. Table 25.20 summarizes the MSS yields of N-heterocyclic amines considered to be
significant tumorigens [Hoffman and Hoffmann (1740, 1741)] plus the assessment of the IARC
(1870) on their tumorigenicity in laboratory animals and humans. Table 25.21 illustrates
precursor relationships, either demonstrated or proposed, between N-containing components
such as the amino acids and proteins and tobacco smoke components. The tabulation of
possible flavorants for tobacco smoking products by Leffingwell et al. (2341) included
contributions to tobacco smoke taste and aroma of 23 amino acids added individually to the
cigarette filler.

During tobacco growth, curing, aging, and/or the smoking process, tobacco sugars may react
with ammonia and/ or amino acids to yield Amadori compounds which, when heated during the
smoking process, will generate a variety of pyrazines [Green et al. (1369)]. Many pyrazines
identified in tobacco smoke are highly flavorful and contribute uniquely to the aroma and taste
not only of tobacco smoke but also of a variety of consumer food products such as coffee, tea,
cocoa, roasted peanuts, and roasted, broiled, or fried meats, poultry, and fish [Maga and Sizer
(2439)].
25.4 TOBACCO
ADDITIVES

25.4.1 ADDITIVES USED IN TOBACCO

PRODUCTION

Much information exists in the tobacco literature on the use and levels of use of various
materials added to tobacco during growth, harvesting, and storage and on such materials that
either remain unchanged on the tobacco as residual material or are chemically altered. These
materials include insecticides, herbicides, fungicides, fumigants, and sucker growth inhibitors
(see Chapter 21). Acceptable use levels of these are prescribed in the United States by
appropriate government agencies. Comments, eg, see Wynder and Hoffmann* (4332), Guthrie
(1457), and Guthrie and Sheets (1460) on the use of pesticides, etc., were published in
increasing numbers, after the mid-1960s when smoke components or classes of components
allegedly responsible for the effects of cigarette smoke in smoke–disease association could not
explain the observed effects at the levels in cigarette smoke. Two types of materials have been
examined in greater detail than most of the others. These are discussed in this section because
there is substantial information on their pyrolysis products, their transfer per se from cigarette
tobacco to its MSS, their degradation during the actual smoking process, and/or their effect as
either transferred or degraded materials on the biological activity. Even in these two cases, no
attempt has been made to include all the available references. These include the following
classes of materials:

• Sucker growth inhibitors: Representative sucker growth inhibitors or suckering agents

include maleic hydrazide, currently used as an alkali metal salt, and the normal,
even-numbered carbon chain saturated alcohols, ranging in carbon chain length from
(C6) 1-hexanol through C12 (1- dodecanol) (4332).
• Pesticides: Particularly those pesticides that are chlorinated, eg, DDT, Aldrin®,
Dieldrin® (4332).

25.4.1.1 Sucker Growth

Inhibitors

The pyrolysis of long-chained saturated alcohols such as 1-docosanol and long-chained

unsaturated alcohols such as phytol and solanesol, known to be naturally occurring components
of tobacco, was discussed previously. One of the most widely used and effective commercial
preparations used for inhibition of sucker growth is “Off-Shoot-T,” a mixture consisting primarily
of the evennumbered straight- chained alcohols 1-hexanol, 1-octanol, 1-decanol, and
1-dodecanol [Collins et al. (25A16)]. Pyrolysis studies by Higman et al. (1644, 1645) with
individual alcohols of “Off-Shoot-T” revealed that much of the alcohol was transferred intact to
the pyrolysate. Much of the remainder was converted to the corresponding alkene; eg,
1-decanol yielded 1-decene. Because of their volatility and low molecular weight, conversion of
the alcohols to PAHs was minimal. Definitive evidence that the alcohol sucker growth inhibitors
added to tobacco did not augment the tumorigenicity of cigarette MSS was provided in the
second set of experimental cigarettes studied in the NCI “Less Hazardous” Cigarette Program
(1330, 2683). The chemical and biological properties of the MSSs from three samples
(hand-suckered tobacco, tobacco treated with the recommended level of alcohol sucker growth
inhibitor, and tobacco treated with 100 times the recommended level) were compared among
themselves and with the Standard Experimental Blend, SEB II. Data obtained are shown in
Table 25.22. Examination of the data indicates that neither the normal use level of the alcohol
nor a use level 100 times normal had any significant adverse effect on the mainstream CSC
properties. The MSS phenol yields were increased from the hand- suckered and both
alcohol-treated tobacco samples, but the increase elevation had no significant effect on the
CSC biological properties. The results were described by Gori (1330) as follows:
No statistically significant differences were observed among Hand-suckered, Fatty
Alcohol-Normal and Fatty Alcohol x 100 Blends (variables 60, 61, and 62).

In the summary report (2683) on the four sets of NCI Tobacco Working Group (TWG)
experimental cigarettes, this statement was expanded:

The fatty alcohol, fatty alcohol x 100, and hand-suckered blends showed no significant
differences among themselves or from the SEB II blend.

It is interesting to note that biological responses (% TBA), ranging from a high of the average of
48% for the four replicate SEB II CSCs to a low of 36% for the CSC from the sample treated
with alcohol at the normal use level, were considered to show “no significant difference.” Maleic
hydrazide, another growth inhibitor used as a suckering agent on tobacco, is used in the United
States as its potassium salt. Prior to 1982, use on tobacco involved application of maleic
hydrazide as its diethanolamine salt. Such use was banned in 1981 by the Environmental
Protection Agency (EPA) (1147) soon after it was reported that tobacco treated with it generated
N-nitrosodiethanolamine (NDELA) during smoking. NDELA was subsequently reported to be a
potent, tissue-specific tumorigen in laboratory animals [see Hoffmann et al. (1696) and
references therein]. Over the years, the pyrolysis of maleic hydrazide has been much studied,
eg, by Patterson et al. (2907), Smith et al. (3728), Harke et al. (1507), and Clough et al.
(25A13). Also studied have been its transfer (estimated at ≤4%) as intact maleic hydrazide from
tobacco to cigarette MSS [Haeberer (1470), Liu and Hoffmann (2383, 2384)] and its generation
of hydrazine during smoking (2385).

However, in his 1979 report, the US Surgeon General (4005) noted that maleic hydrazide was
not a significant precursor of either hydrazine or 1,1-dimethylhydrazine in cigarette smoke.
Smith et al. (3728) reported that the pyrolysis of maleic hydrazide at 600°C yielded CO2 (24%),
CO (2%), HCN (3%), NH3 (9%), N2 (3%), hydrazine (trace), and a black residue (50%),
structure unknown, whose empirical formula was C15H15N5O2. In the fourth set of
experimental cigarettes in the NCI's “Less Hazardous” Cigarette Program, the chemical and
biological (mouse-skin-painting bioassay) properties of mainstream CSC from a
“pesticide”-treated tobacco cigarette were compared to those of the CSC from a control
cigarette, SEB IV (1333, 2683). Gori (1333) listed the pesticides, sucker growth inhibitors, etc.,
used and described the chemical analyses and bioassays of the pesticide-treated and control
tobaccos and their MSSs. Among the additives used in the treatment of the “pesticide”-treated
tobacco were maleic hydrazide (MH-30), a 10- carbon alcohol (“Contak”), and DDT. Because
both maleic hydrazide and DDT were on the tobacco, the chemical and biological results from
the MSSs from these samples are discussed later.

25.4.1.2
Pesticides

As mentioned in earlier chapters, the biological response observed in mice skin painted with
CSC or its fractions cannot be explained on the basis of the identified components and their
levels in the CSC. In an attempt to define the biological response, Wynder and Hoffmann
fractionated CSC and determined that the major part of the tumorigenicity that could be
accounted for (only a few percent) arose from a PAH- rich fraction designated as Fraction B
[Wynder and Hoffmann (4332, 4342), Hoffmann and Wynder (1798, 1800)]. In addition to 39
PAHs, totally or partially identified, among which were several known to be mouse-skin
tumorigens, Fraction B contained 27 N-heterocyclic compounds (indoles, carbazoles, acridans),
5 O-heterocyclic compounds (dibenzofurans), and 6 chlorinated compounds that were either
insecticides (DDD, DDT) or their chlorinated derivatives (trans4,4′-dichlorostilbene) [Hoffmann
and
Wynder (1800)]. According to Hoffmann and Wynder (1800), trans-4,4′dichlorostilbene (DCS) is
one of the major pyrolysis products of the most important tobacco insecticides DDT and DDD.
They also stated:

DCS is neither a complete carcinogen nor a tumor initiator, nor a tumor promoter, but the
DCS (0.3%) can accelerate significantly the tumorigenicity of a BaP solution (0.003%)
when both agents are applied concurrently.​

However, it should be noted that the use in tobacco culture of chlorinated insecticides such as
DDD and DDT in the United States was discontinued in the late 1960s. For example, between
1968 and 1974, the residual DDT levels per gram of US flue-cured tobacco decreased rapidly
and substantially (over 200- fold) as follows: 1968, 52 μg/g; 1970, 6 μg/g; 1974, 0.23 μg/g
[USPHS (4005), IARC (1870)]. From 1967 through 1973, the organochlorine-containing
pesticides such as DDT and TDE were subjected to detailed examination not only for their
contribution to cigarette MSS composition by direct transfer and/or degradation to simpler
compounds during the smoking process but also to the composition of their pyrolysates.
Investigators involved included Nesemann et al. (1968) from BAT (West Germany), Hoffmann et
al. (1756, 1767) from the Sloan Kettering Institute and American Health Foundation, Carpenter
and Frost (606) from Carreras Tobacco, Chopra et al. from North Carolina A&T [Chopra and
Osborne (709, 25A12), Chopra and Domanski (707), Chopra et al. (708), Chopra and
Thekkekandam (713, 714)], and Kennedy et al. (25A36) from Mississippi State.

Chopra and Osborne (709) initially studied the pyrolysis of p,p′-DDT to identify degradation
products. They compared the pyrolysis data with those from actual smoking studies and
commented on the differences observed: ​There are two differences in the pyrolysis of DDT

reported earlier and the degradation of DDT in tobacco

​ smokes: the concentration of DDT is
much greater in the former and hydrogen in the latter. The DDT degradation products as we
have found are as would be expected from the difference in the reaction conditions. It is thus
possible to predict the fate of a pesticide in tobacco smoke by studying its pyrolysis pattern.
 Also these investigations show that at the combustion zone hydrogen plays a very important
role in the reactions taking place.

Chopra et al. employed these pyrolysis data to identify the transfer and degradation products
generated from pesticidetreated tobaccos during the smoking process [Chopra and Domanski
(707), Chopra et al. (708), Chopra and Osborne (25A12), Chopra and Thekkekandam (713,
714)]. The results reported indicated the effect of added pesticides on MSS composition
primarily in terms of the transfer of intact pesticides from tobacco to smoke or the products
generated from them during the smoking process. In the NCI study of the fourth set of cigarettes
(1333, 2683), the effect of added pesticides on chemical and biological properties (mouse-skin
painting) was examined. As noted previously, the “pesticides” included the sucker growth
inhibitors maleic hydrazide (MH-30) and “Contak®” (1-decanol) in addition to DDT (1333). Other
compounds present in the mix used in the tobacco treatment according to government-
approved procedures and treatment levels included Lorsban®, Dylox®, Enide®, Lannate®, and
Carbaryl®. According to Smith et al. (3727), pyrolysis of Carbaryl® (methyl carbamic acid,
1-naphthyl ester) gave three major products in the pyrolysate: unchanged Carbaryl (»40%),
1-naphthol, and methyl isocyanate. Some results from the NCI study of the MSS from the
“pesticide”-treated tobacco are shown in Table 25.23. The conclusions were [see p. 29 in
(1333)] as follows:
Pesticide-Free and Pesticide-Treated Tobaccos: Two cigarettes tested in the fourth
experiment were made from tobacco grown [in] Prince Edward Island (PEI) [Canada].
One of the tobaccos was pesticide-free and the other was pesticide-treated... There are
no statistically significant differences among the [probability of survival] values at either
dose level... Relative condensate yields from these cigarettes are presented... These
yields confirm the [probability of survival] values, namely: there is no clear cut difference
between the pesticide-free and pesticide-treated tobaccos.

It was also noted in the summary (2683) of the NCI 10-year “Less Hazardous” Cigarette
Program:

No significant differences were observed between cigarettes made from

pesticide-treated tobacco leaves and pesticidefree tobacco leaves.

These findings, at least with respect to the maleic hydrazide added, are in agreement with the
comments of Chopra (704) in his theoretical discussion of the relationships among pyrolysis,
maleic hydrazide, the actual smoking of tobacco treated with maleic hydrazide, and B[a]P:

Evidence and data so far available on maleic hydrazide are not sufficient to suggest the
MH [maleic hydrazide] is a health hazard to the smoker... Thus far it is reasonable to
assume that there is not enough data or justification to consider the use of MH as a
health hazard to the smoker.

In the late 1970s, two excellent reviews on reagents used to treat tobacco and their effects on
tobacco chemistry were authored by Steffens (3911a) and by Sheets and Leidy (3634). The
latter review included considerable information on the effect of such compounds added to
tobacco on MSS composition. Sheets and Leidy (3634) summarized the data which indicated
the gradual decline in the levels on tobacco of insecticides such as DDT following the
government's proscription of their use on tobacco (and other crops) in the United States. Their
summary is similar to that provided earlier by Guthrie (1457) on the gradual decline of the
tobacco arsenic level following the discontinuance in 1952 of arsenate use on tobacco. By 1968,
the arsenic level in US tobacco had decreased from a 1951 level of ≈50 μg/g of tobacco (dry
weight) to 0.5–1.0 μg/g [USPHS (4005) 1979d; IARC (1870)]. In 1975, Griffin et al. (1391)
reported arsenic values of 0.5–0.9 μg/g for US tobaccos. A pesticide used to control cigarette
beetles in stored tobaccos and tobacco products was methoprene (Altosid®) or in the
formulation Kabat®. The use of the well-studied methoprene
(5-isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) escalated markedly in the
1990s. Methoprene is also acceptable for controlling pests on various foodstuffs as well as
mosquito larvae in water supplies. Using radiolabeled methoprene in a cigarette, Frisch et al.
(1242, 1243) reported that 38.2%–39.4% of the activity was found in the MSS, 52.3%–52.4% in
the sidestream smoke (SSS), and 8.1%–8.4% in the 23 mm butt. Of the activity in the MSS,
96.8% was due to methoprene transferred from the treated tobacco. Of 1.3% total activity found
in mainstream vapor phase, 86% consisted of radioactive CO plus CO2; the remaining 14% of
the activity was distributed among 10 vapor-phase components, all normally found as
vapor-phase components of cigarette MSS.

25.4.2 ADDITIVES USED IN CIGIRETTE

MANUFACTURE

The pyrolysate compositions from various tobacco additives, including casing materials and
humectants, and their effect on cigarette MSS composition and properties were described by
Roberts et al. (3225, 3226) and by Schumacher (3551). In this section, additional data are
discussed.
25.4.2.1 Casing Materials (Sugars, Cocoa,
Licorice)

Casing materials used in cigarette products in the United States are licorice, cocoa, and the
sugars— including the invert sugars (glucose plus levulose) and sucrose. While they did not
discuss the topic in their 1964 review (4319), Wynder and Hoffmann (4332) did discuss the
possible effects on cigarette MSS properties of inclusion of casing materials in the tobacco filler
with particular emphasis on licorice because of its glycyrrhizin content. Glycyrrhizin, the
potassium and calcium salt of glycyrrhizic acid, is polycyclic with five cyclohexane rings in the
picene configuration. (Figure 25.2). Picene is a PAH identified in several pyrolysates by Badger
et al. (142) and Kröller (2195) and in CSC by Snook et al. (3756).

According to an assertion by Wynder and Hoffmann (4332), glycyrrhizin in licorice added to

tobacco could be a precursor of PAHs in smoke. In an ill-defined experiment, Hoffmann et al.
(1766) compared the B[a]P yield in the MSS from pipe tobacco (containing 30% casing
materials, including licorice [level unspecified]) smoked in a pipe (2 puff/min) with the B[a]P level
in the MSS from cigarette tobacco (no licorice added) smoked identically, ie, in a pipe. The
B[a]P levels were 27 and 10.5 μg/100 g of tobacco smoked, respectively. Later, Hoffmann and
Rathkamp (1754) stated that the pyrolysis of licorice yielded PAHs. This finding was
subsequently confirmed by Green and Best (1356, 1357) whose data on B[a]P generated during
identical pyrolyses of licorice and flue-cured tobacco are summarized in Table 25.24. They also
identified 35 other compounds in the licorice pyrolysate [Green and Best (1356, 1357)], all of
which had previously been identified in cigarette MSS. Ten of the licorice pyrolysate
components were phenols; four were dimethyl- or trimethylnaphthalenes. Many of the
compounds identified in licorice are the same as those identified in tobacco (3551, 3555). Thus,
there will be similarities in their contributions to the composition of either pyrolysates from
licorice vs. tobacco or the MSSs from a licorice-containing vs. a licorice-free tobacco blend.
Differences will be reflected by the components unique to the material being investigated, eg,
glycyrrhizin in licorice, theobromine in cocoa, and nicotine in tobacco. As noted by Schumacher
et al. (3555), 172 (83%) of the 209 components identified in licorice by 1981 had also been
identified in tobacco and/or tobacco smoke. Later, a similar situation between cocoa
composition and tobacco/tobacco smoke compositions will be discussed. Sakuma et al. (25A62)
reported the pyrolysis products from several naturally occurring polyphenols (chlorogenic acid,
rutin). Rutin is a major polyphenol in both licorice and different tobacco types. It and chlorogenic
acid yield substantial levels of catechol and substituted catechols on pyrolysis. All the identified
volatile pyrolysis products from rutin have been identified in cigarette MSS. In the late 1970s,
Harllee and Leffingwell (1512, 1513) cataloged cocoa components identified to that date with
particular emphasis on the volatile, flavorful components common to cocoa and tobacco or its
smoke. Both tobacco and tobacco smoke as well as cocoa contain numerous fatty acid
triglycerides (1512) and many of the same fatty acids (1512). At least 19 amino acids are
common to cocoa and tobacco (1512). Of 352 volatile components identified in cocoa by 1979,
209 (59%) had also been identified in tobacco and tobacco smoke (1513). From his pyrolysis
study on cocoa, Schlotzhauer (3447) reported that his results suggested the following:

Cocoa powder added as tobacco flavorant would not significantly increase the phenol
yield of smoke, but may affect the higher fatty acid content...

In the NCI study of the third set of experimental cigarettes (1332), the cigarette MSS
composition and biological properties (mouse-skin-painting studies) of four cigarette samples
were compared. These included three samples to each of which had been added individually a
specified amount of glycerol (Code No. 80), sugar (Code No. 81), and cocoa (Code No. 82).
The fourth sample was a control (Code No. 83) to
which none of these casing materials/humectants had been added (1332, 2683). The results are
summarized in Table 25.25 together with data from four Standard Experimental Blend III
samples (SEB III, Code Nos. 72–75), the controls for the third set of experimental cigarettes.
Variations in the analytical and biological data among these four control samples (Code Nos.
72–75) raised questions about any attempt to compare on a one-to-one basis the data from
individual samples, eg, the cocoa-treated sample (Code No. 82) vs. its control (Code No. 83),
the sugar-treated sample (Code No. 81) vs. its control (Code No. 83), etc. Additional comments
will be made about these data in the following section where humectants are discussed. The
phenol data (Code No. 82 vs. Code No. 83) in Table 25.25 indicate the prediction by
Schlotzhauer (3447) appears to be valid: Inclusion of nominal levels of cocoa in the cigarette
blend produced little change in the MSS phenol yield. Addition of 1% cocoa (Code No. 82 vs.
Code No. 83) increased the phenol yield [mg/g of CSC] by 0.13 mg/g. This is about a 3%
increase relative to Cigarette Code No. 83, well within the experimental error of the phenol
determination.

25.4.2.2 Humectants (Glycerol, Propylene

Glycol)

Humectants (glycerol, propylene glycol) are added to the tobacco blend to diminish the rate of
postcigarette manufacture moisture loss. Cigarettes are usually manufactured with the blend at
a 12% moisture content. As the cigarette loses moisture, ie, becomes “dry” during
transportation, shelf storage, its yield of smoke components changes adversely with increased
yields not only of particulatephase entities such as “tar” and nicotine but also vaporphase
components such as the aldehydes (acetaldehyde, acrolein) and ketones (acetone, methyl vinyl
ketone) [Green et al. (1364)]. These changes usually are perceived by the consumer to be
detrimental and unacceptable [Townsend (25A76)]. The pyrolysis of humectants, including
glycerol and propylene glycol, was studied in the mid-1960s by Doihara et al. (1023, 1024) and
Kröller (2192, 2195, 2196). Kröller examined the pyrolysates from nine humectants (including
glycerol and propylene glycol, the most commonly used in the United States) for the yields of
B[a]P and other PAHs generated during the pyrolysis. His B[a]P data are summarized in Table
25.26. In the early studies of methods to control cigarette MSS composition and yield,
particularly with regard to PAHs, Bentley and Burgan (286) asserted addition of glycerol at the
3% level to the tobacco blend substantially decreased (by as much as ≈60%) the B[a]P yield
when the CSCs from glycerol-treated cigarettes vs. untreated tobacco were compared. Their
finding was challenged by Wynder and Hoffmann (4332). Subsequently, with a reproducible
analytical method for B[a]P determination, Scherbak et al. (3440) and de Souza and Scherbak
(953) reported that such levels of added glycerol did not have the dramatic effect claimed by
Bentley and Burgan on PAHs (or B[a]P) generation during smoking. Scherbak et al. (3440)
reported: ​Addition of glycerol to flue-cured tobacco up to the 6% level does not modify the

formation of 3,4-
benzpyrene or smoke
particulate.

The effect of glycerol added to the cigarette blend on MSS composition and properties was
studied by the NCI TWG in the third set of experimental cigarettes (1332, 2683). The results
were summarized in Table 25.26. At the glycerol level (nearly 3%) used in the glycerol-treated
sample (Code No. 80), it would be expected that the total particulate matter (TPM) would
contain sufficient transferred glycerol to dilute other components by about 10%–12% [Greene et
al. (1382), Laurene et al. (2300), Wynder and Hoffmann (4332), Hege (1603)]. This was not
observed with B[a]A and B[a]P yields but was with the phenol yield [cf. data in Table 25.26 for
samples of Code Nos. 80 and 83]. The effect of transfer of humectants from the tobacco blend
to smoke plus their dilution of the products formed during the cigarette smoking process
affects the biological properties (mutagenicity in the Ames bioassay with S. typhimurium) of the
MSS particulate matter. In a detailed study of the effect of various casing ingredients (sugars,
cocoa, humectants) on smoke chemistry, Baker et al. (174c) determined the yields of various
“Hoffmann analytes” when such ingredients were added in various mixtures. They concluded:

Many of the casing ingredient mixtures either had no statistically significant effect on the
level of analytes investigated in smoke relative to a control cigarette or the produced
decreases of up to 44% in some case.

Sugars did increase the yield in MSS of

formaldehyde.

25.5 CIGARETTE CONSTRUCTION MATERIALS (PAPER,

ADHESIVES, ETC.)

Interest in cigarette MSS components possibly responsible for the epidemiological

cigarette smoking–lung cancer association and the biological response observed in mice skin
painted with massive CSC doses led to studies of the contribution of cigarette paper to MSS
composition, primarily because as a cigarette construction factor, other than the tobacco blend,
it contributed substantially (6%–7%) to the weight of a 1.0 g cigarette. In 1954, Cooper and
Lindsey (819), based on fragmentary UV data, reported the presence of several PAHs, including
B[a]P, in a “tar” obtained by burning cigarette paper in bulk. Similar findings on PAHs (and
B[a]P) in the combustion products of cigarette paper, tobacco, and cigarettes were reported by
Lefemine et al. (2335), Cardon et al. (594), and Alvord and Cardon (57). They also proposed an
additive (ammonium sulfamate) for paper and/or tobacco to reduce pyrogenesis of PAHs. The
ammonium sulfamate efficacy in decreasing B[a]P production in a burning cigarette was
discussed by Wynder and Hoffmann [see pp. 521–523, 528 in (4322)] who noted that the
additive gave discordant results in different investigations [no significant B[a]P reduction
reported by Bentley and Burgan (286) or Pyriki et al. (3046) vs. substantial B[a]P reduction
reported by Alvord and Cardon (57), Lindsey et al. (2370), and Candeli et al. (589)]. Whether
cigarette paper contributed significantly to the PAHs in cigarette MSS was finally resolved by
Wright (4281) who reported that PAHs (and particularly B[a]P) were indeed generated when
cigarette paper was burned in bulk or pyrolyzed, but when it was burned in a cylindrical
configuration such as that encountered around the cigarette tobacco rod, the yields of PAHs
(particularly B[a]P) were insignificant. Between 1963 and 1966, Kröller (2184–2195, 2200, 2203)
reported the pyrolysis products from cigarette components permitted in cigarette fabrication in
Germany. He estimated the amount of B[a]P generated by pyrolysis of each material at 700°C
in air. In addition to tobacco itself (2191), the materials he studied included cellulose, starch,
and a number of humectants, adhesives, and dyes consumed during the actual cigarette
smoking process. Kröller (2191, 2192) asserted his pyrolysis and the actual cigarette smoking
process were qualitatively and quantitatively identical processes. Kröller's opinion of the
equivalence of the fate of a material on pyrolysis in an inert atmosphere vs. its fate in the
tobacco rod of a smoked cigarette parallels that of Wynder and Hoffmann (4332). In addition to
phenanthrene, 4,5-methylenephenanthrene, and fluoranthene, Kröller reported the identification
of four potent PAH mouse-skin tumorigens in his tobacco pyrolysate: DMB[a] A, 3-
methylcholanthrene (now known as 1,2-dihydro-3methylbenz[j]aceanthrylene), B[a]P, and
DB[a,h]A. Despite their agreement with Kröller (2191, 2192) on the equivalence of the fate of a
material in an inert atmosphere pyrolysis and the smoking process, Wynder and Hoffmann were
critical of several Kröller's findings. For example, Wynder and Hoffmann (4332) questioned
Kröller's identification of the methyl derivatives (DMB[a]A, 30-methylcholanthrene) in his
pyrolysates and smoke samples. However, a methylbenz[a] anthracene had been identified in
CSC by Rodgman and Cook (3273) in the late 1950s, and
numerous methyl- and dimethylbenz[a]anthracenes were subsequently reported in CSC in the
late 1970s by Snook et al. (3756, 3757). Table 25.27, adapted from Kröller (2195), summarizes
his data on the amounts of B[a]P generated during the pyrolysis of tobacco and numerous
components used in cigarette fabrication. More recently, several studies on the pyrolysis of
various adhesives either used or proposed for use as seam pastes in cigarette fabrication were
conducted by Best (25A06, 25A07). He reported that, in contrast to a starch pyrolysate, the
pyrolysate from polyvinyl acetate showed high levels of acetic acid and B[a]P. BEST also
examined the pyrolysates from a variety of proposed new cigarette papers (25A04, 25A05,
25A08). In 1967, Wynder and Hoffmann [see pp. 350–351 in (4332)] noted that pyrolysis data
on cigarette components should be considered carefully:

Certain casing agents, saucing materials, and humectants are widely used in the
manufacture of tobacco products. For all we know at this time, it is certainly a possibility
that PAH may also be formed from these agents. One should keep in mind, however,
that only small amounts of these materials are used for most smoking products.

Their latter statement would also apply to cigarette fabrication materials consumed during the
smoking process such as the adhesives used on the cigarette paper seam and printing inks on
the paper. The data from Kröller (2195) on the B[a]P yields generated during cellulose and
starch pyrolysis may be compared with those of Gilbert and Lindsey (1289), see Table 25.28.
This provides an excellent example of the effect of changing pyrolysis conditions (pyrolysis at
650°C in air vs. pyrolysis at 700°C in N2) when two different materials are considered. The
different pyrolysis conditions give a ratio of 9.75 (0.78/0.080) for the B[a]P yields from cellulose,
but a ratio of 2.43 (0.17/0.070) for the B[a]P yields from starch. Inclusion of cellulose in the
Kröller studies was because it constitutes the major part of cigarette paper, much of which is
consumed during the smoking process. It was not included because cellulose, as wood pulp, is
sometimes added by some manufacturers to their reconstituted tobacco sheet (RTS) to improve
integrity and reduce fragmentation. The effect of cellulose added to cigarette filler on cigarette
MSS composition and properties in a mouse-skin-painting bioassay was examined in the
mid-1970s in the NCI study of the first set of cigarettes (1329, 2683). The results are poorly
defined because the cellulose was added (as wood pulp) at a 7.5% level to fillers made from the
Standard Experimental Blend (SEB I) reconstituted into sheet material at three different
densities.