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BATCo document for PFSFC 1 March 1999
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B.A.T. CONFIDENTIAL - CATEGOR.Y I: MINNESOTA TOBACCO LITIGATION
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BATCo document for PFSFC 1 March 1999
COPY NO.~
IMPERIAL TOBACCO ~TD.
RESEARCH & DEVELOPMENT DTVTSTON
MONTREAL
RESTRICTED
PROGRESS RE?ORT
JANUARY 1994 - JUNE 1994
B.A.T. CONFIDENTIAL - CATEGOR.Y I: MINNESOTA TOBACCO LITIGATION
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WORK A. -'-LEA 02
EXPLORATORY PRODUCT RESEARCH
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RECORD TYPE :
SUB TYPE:
SECURITY CODE:
FUNDING BODY :
ORGAN I ZAT I ON:
GROUP NUMBER:
LOCAL PROJECT NUMBER(S):
PROJECT TITLE:
PERSON RESPONSIBLE:
EFFORT:
PROJECT DESCRIPTION :
SCOPE:
DEPTH."
FUNCTION:
OBJECTIVE :
~USTER:
DATE, REVI-~W WRITTEN:
REVIEW TITLE:
REVIEW TZ.XT:
$TL CANADA
578
T-6431;T-771!-2;T-6542;T-7344;T-6968;T-~549
Day
PORTER, A.
5.7 = 1994
This projecu £nvolves ~he development of a
uohacco combusuion cigarette 0£ convennional
appearance bur which produces smoke with low
speclfic mu~agenicity and low concentraulons of
PAH, phenols, vapour phase aldehydes, HC~ and
CO. This p=oduc~ will be designed uo provide
accep=able uasue and nicotine, while producing
:educed levels of sideeuream smoke oompued
wi:~ convenuional products. The principal
su:auegy foe achieving reduo~io8 of ~he
pa:~iculauo s~oke noxae is by ~obacco
modification using solvent exuraction (T-6431),
enzyme/microblal ~rea~men= (T-7711-2), and
reccns~i~u~ion (T-6S42). Emphasis will also be
placed on re~aining ~obaoco flavou: ¢omponenus
during ~he various modification processes.
Vapou: phase noxae will be reduced by
convenuional ¢!gareuue componenus such as
abso:~en= fil~eEs and venuilauion (T-7344).
Ana!y~ical me,hod development |peoifi¢ally for
DAY is also included in this proje¢~ (T-6968,
T-5~49). Successful developments Of low
sidesuream produc~s via prcjecU VISA will be
incarporated inUo DAY as appropriaue.
GROUP BENEFIT
FUNDAMENTAL
GENERAL
ALTERNATZVE
PRODUCT RES/DEV
July 1994
Day
~cbacco Frag~ionan~on ¢De Grand=r~, Y.~
(T-6431}
The effec~ of the extEa¢~icn prccedureon ~he ~=bacco tasue level and on the
impact of the ~oke was investigaued using a new blend. The ~amina blend of
JPS, v-23, was chosen since i~ s~owed high ~asue level and high impac~
compared ~o the previously used blend, V-Og, which had suEprising!y low ~asts
level and impact.
Three samples were prepa:ed using ~he ~echni~e desc:ibed in a previous
Progress Repo~-u (January '$3 - July '93). The UEeaued solubles were added-
back s~ 45% (cigarette #418), 5~.59 (cigaren:8 #397) and 509 (cigarette #257)
of ~he ta=al ~obacco wei~h:. A fou::h cigaEe~Ue was made wi~h ~hs uriC:eared
lamina blend (#907). The ::bacco and smoke chemist:y are presenusd in Table
#I.
B.A.T. CONFIDENTIAL - CATEGORY I: ~[INNESOTA TOBACCO LITIGATION
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TABLE I: ,,P~YSZC,~ AND SMOKZNG RESULTS
Leaf NLc. (%)
Red. Sugars it)
CLgu. P.O. (t)
Puff Number
TPM (rag/c~gu)
(mg/puff)
TAR (mg/cigu)
(mg/puff)
WATZR (mg/clg~)
(n~/puff)
NIC. (mq/ciq~)
{mg/puff)
co (mg/cicj~.)
(mg/puff)
CO/T
T:NIC.
~907
~.82
'.'.6
"31
-:.8
~3.25
1.37
IT.93
I #415
1.95
17.2
128
9.1
18.47
2.03
14.73
#397
1.95
17.3
131
9.4
18.69
1.99
14.76
1.83 1.62
2.58
0.28
"-.83 1.15
C.19 0.13
I~.83
.'.19
1.05
~.8
1.57
2.75
0.29
1.18
0.13
16.32 16.98
1.79 1.81
I.ii 1.15
12.8 12.5
#257
2.51
22.3
iii
9.9
20.48
2.07
15.41
1.56
3.47
0.35
1.61
0.16
18.09
1.83
1.17
9.6
The hes~ nicotine recoveEywas ob=ai~sd *
.=: cigars~ue #2S7 wL~h ~he highes=
addition of solubles (60%). All clgareU=ss were smoked for phenols,
aldehydes, benzo[a]pyrene, HCN and Ares s~acific activity,. The resul~s are
presented £n Table 2.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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TABLE 2: SMOKE ANALYSTS
Smoke Componen=s
Puff ~.~'~er
TAR (mg/cL~=)
PHEXCLS (#g/=i~=)
Ratio (gg phenols/mg tar)
ALDEHYDES (~g/ci~u)
Ratio (~g a!dehydes/mg tar)
B(a)? (ng/cigul
Ratio (ng B(a)P/mg ~ar)
HCN (gg/cig~)
Ratio (pg HCt;/mg tar)
Spec. Activit7
I #907
9.8
17.9
301
16.8
1415
80
15
0.84
303
16.9
1.3
#418
9.1
14.7
137
9.3
1514
103
13
0.86
101
6.6
9.4
14.8
i 144
L 9.7
!521
14
0.95
7.0
I #257
9.9
lq.4
174
11.3
IS70
102
15
0.97
149
9.7
0.62 ~ 0.67 0.84
i
The deliveries of =he three trea=ed cigarettes (#418, #397, #257) arm lower
nhan the untreated =he, #907, ex=ep= for ~he aldehyde deliveries.
Subjectively cigare==e #257 was superior ~o cigare=~es #413 ~n.d #397. Moz~
specifically =igareu=e #257 had an average impact, some i==ita=ion, an averse
quality taste wi:h an average ~as~e level, an off-~asue cn ~he fi~s~ puff and
accep=a~!e mechanics. Clgare~=me #418 and #397 had highs= i==i:a~ion, a l~r
impao~ and a lower caste quali~y than cigarette #257.
The effect of ~he ex=mac=ion procedure on ~he =obacco ~as=e an~ on =he ~pa=
of ~he smoke is also being investigated using various ~o~a::o treatments. The
following to, acre samples are presently being prepared: a) V-23 ~oba=cm
e~Eac~ad wi~h wat~= only ~ha~ will be sprayed-hack wi~h a c=noen~ra=m~
solu=ion of uncreated solubles; b) v-23 tobacco ex~:ao~s= wi:h wa~er,
followed by an enz.vn..e/deuergen= extras:ion of the ~obac=: resilue ~hau will be
sprayed-back wi:h a concen=ra~ed solution of uncreated solublss; c) V-23
~obacco extracted wluh wa~er, followed by an enzyme/deter;ant extraction of
~he ~obaoco residue that will he spraye~-haok with a concsn=r~usd solution of
soluhles :rma~sd with bennonite only; d) V-23 ~o~acco ex=rac=sd with water,
followed hy an enzyme/detergent extraction of ~he ~ohaoco =esi~ue ~han wall be
sprayed-back wi~h a concentrated solution of solubles ~rsa=ed wi~h bentonite
only; e) V-23 ~obac:o extracted winh water, followed by an enzyme/de=ergen~
extraction of nhe ~:bacco residue tha~ will be sprayed-back with a
concentrated solution of solubles ~rea~ed winh pvpp only. This ~xperimen~
will ex~=ine the effects of each s~ep of ~he process separa=e!?. Cigarettes
made from these ~obacco samples wall be physically, chemically and
subjectively assessed.
Fiavour Re~ease (T-5175)
Tobacco flavou=s and volatile$ were exu=ac~ed fr=m V-23 bi~nd =y s~sam
dls~illa=Ion and ine== gas dls~illa~i=n.
Zn ~he s=eam dis=ills=ion ~echnique 100 g of cur or ground =c~a::o are
dispersed in one li=ar of wooer and heated =o =he boiling ;:in=. The
dis~illa:e is co!letted in an organic s¢Iven= and :he flav=u=s recovered after
evaporauion of ~he solvent. Wi=h ~he !nero gas disni!laui:n us:~ni~4e i00 g
of grounl :o~ac=o are placed in a one-lluer flask. The :==~c:= is heated to
about I~=C. An iner= gas flow, 100 ¢o,'=in. of No, is pas~e~ uhc=u~ :he
B.A.T. CONFIDENTIAL - CATEGORY I: ~IrN'NESOTA TOBACCO LITIGATION
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~obacco bed during hea~ing and the vola~iles are collec=ed in a c=ld ~ap a~
-80"C. The vola~lles are then recuperated by a liquid/!iquid extraction. In
general the yield of the flavours obnained varied between 0.57 and 0.15% of
~he ~obacco weight. The flavours were dispersed £n ethanol and £njectsd, au
0.I and 0.2% of the tobacco weight, in Day 9=ototypes (#418, #397 and #257)
and subje~ivl!y smoked. The flavours obtained by steam d£suillation were
subJeculvel7 supl=Lor UO ~hose obtained by the ine~ gas dis:illauion. The
steam dis~illed fiavours also improved the subjective properties oE the smoke
¢ompar~ to ~he same prototype without flavours. The most no~iceahle
Improvement was observed in prototype 257.
The flavours were also adsaTbed on sepiolite, an inorganic su~por~, at a
weight e cuivalenu to 0.i and 0.2% of the tobacco rod weight. The ~reaued
sepio~ite was inse~ed into ~he filter of the prototype cigarette and
subjectively smoked. Once again the flavours obtained by sulam dLsuillatlon
wore subjectively superior for the three prototypes smoked.
Finally =he injected pEc=o~ypes were considered superior =o chl prototypes
made wi=h the f!avoured sspiolite.
B.A.T. CONFIDENTIAL - CATEGORY I: MrNNESOTA TOBACCO LITIGATION
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RE:DRD TYPE:
SU3 TYPE:
SE=~RZTY CODE:
r=XDING 8ODY:
ORGANIZATION:
GROUP NUMBER:
LOCAL PROJECT NUMbER(S):
PROJECT TITLE:
PERSON RESPO~SZBLE:
EFTORT:
P~0JECT DESCRIPT=GN:
SCOPE :
DE.~TH :
F.=.~CT I 0N :
Oe.'~CTIVE :
C'_'~'STER :
DA~E REVIEW S~ITTZ:I:
REVIEW TITLE:
RZVZEW TEXT:
ITL CANADA
416
T-7711-2
Enziaue/Micr:bial Treatmenu =f Tobacco
BZL~MORIA, M.H.
0.S-1994
This study f~ouses on ~he hydrolysis and
removal of uabacco polymeric maumrials such as
proteins, pec:ins, etc. and uheiE degradaUlon
produc~s using enzymes and/or microorganisms.
GROUP RELEV;~NT
FUNDAMENTAL
GENERAL
ALTERNATIVE
PRODUCT RES/DEV
July 1994
Enzyme/Micr=Dial Treatmenu cf Toba=co
1. Enzvman&c PrO~e~ Remove! from Tobacco in
~resence of Oruanic Solvents: In ~he lasu semi-annual r c r we
• o u showed ~hau
As=er~illus o.--:zae proueaee (Sigma, Type XX:II) could sc!uhi!ize flue-~ure~
tobacco prouein in the presence of aceuone, e~han~l and methanol. These
experiments were c=nducusd au ambienu temperature using a r~tary shaker and
the incubations were carried ou~ for 18 hours. We have now repea~ =his
euudy using a ~emperauure of 40eC. The an=lane treatmen: au this tempe=auure
was carried out for jusu 4 hours, and ~he results ob=ained presented in Table
!. From this ~ab!e i~ will be seen ~hau presence of 10% acetone or e~hanol
had liu~le effecu on uhe activity of uhis pro~ease and uhau protein
ec!ubilizaticn was =ompar~le to uha~ in a to~ally aquenue environmen=. Use
cf 20% aceUone or ethanol reduced protein sclubi!izaUicn only sligh~!y. It
should be polnued out ~ha~ signifi=ant amounts of prouein ware sclubilized at
40"O in the absence of the p:oteaee as compared to that $olubilized in the
earlier suudy carried out an am~ien~ ~empera~ure.
PROTEIN REMOVAL FROM FLUE-CURED TO~Cq~ By ~UNGAL PROT~AS~
IN T~Z P~ESENCE OF ORGANIC SOLVENTS
SOLVENT
WATER
ALKALZ - O.OlM
IC%
2C%
10%
20%
- 85A as suandar¢.
PROTE.'N CONTENT - MG/G~' DWB~
TR~ATME:;T
CONTROL - NO PROTEASE
100.50
90.75 (i0)
84.40 (16)
87.63 (12)
88.38 (12)
77.75 (23)
PROTEASE
26.53 (74)
33.13 (67)
43.75 (56)
35.88 (64)
48.25 {52)
T.~s samples were =rea~ed with pro~ease (5 mG/gma ~ohacc=).cr ~ 4 hours in an
environmen~a! shaker at 40"C.
T.h~ numbers in brackets a • per can= redu=--lDns as compared --= the wa e
r u .-
C3.'. "i.-0 ~..
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2.
Enz-n~.a~i= P~c~e£n Removal from Differs== Tc~a=co Tv~es:
While uhe prouei~ pEesen= in flue-cured tobacco is readily solubLlized by
A. c~v~ae pro:ease au 5 mg/~ Uobac:o, uhe p:o~e£n in dark, air-cured,
fe~enuec =obacco Eesisued solubilizaui=n at uhis enzv=..e concenurauion.
Consequenuly, we decided ~o increase the pr==aase con~enurauion ~wo- and
four-f=Id uo deuermine if protean An ~his dark, fermen:sd Uobacc= could
now be Eende=ed soluble. From uhe resulUs presenued in Table 2, i~ will
be seen ~hau while up ~o 70% of uhe pr=uein in flue-cured uoba¢¢o and 40%
of ~he prcuein £n o=ienual uobacc= was removed, only a negllgible amount
was solubilized from Uhe dark, fs:menu~d ~ubacco even au ~heee high enzyme
concenura=icns.
TAaLE 2
PROTE-TN SOLUBI~T2AT~O~ ~ D~FF~E.'q~ ~OBACCO ~'YP~R
TOBACCO TYPE AND
TRZATHENT TIME
HOURS
."~.LTE-CURED
2
4
6
2
4
6
DARX. AZR-CURED
FE-~ENTED
2
4
6
T IgG as suandArd.
~ROTE=:Z CONTZNT - MG/GM DWB~
*E:Z~YME TREATF~T AT
WATER
CONTRCL i0 MG/GH 20 MG/GM
75.4
76.~
73.3
104.6
102.0
109.4
147.3
143.5
146.3
25.4 (66)
24.8 (68)
23.1 (6a)
76.5 (27)
60.5 (41)
67.6 (3~)
Z3~.6 (7)
134.0 (7]
135.0 IS]
23.S (69)
23.0 (70)
23.6 (68)
7~.0 (28)
6~.0 (35)
65.1 [40)
131.8 (11)
132.1 (8)
127.0 (13)
• ore=ease, urea=menu a= 1~ mg and 20 mq/~-.~. Uo=acco for up to 6 hours £n an
enviro~.~enua! shaker au 40°C.
The ~u~bers in bEackeus are per ¢e~ reductions as compared to wa~er
C
L+~.
P~3
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B.A.T. CONFIDENTIAL - CATEGORY" I: ~|INNESOTA TOBACCO LITIGATION
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Nex=, ~e ex=a=ded =heso s=udLes =o include burley, dark, ai=-cursd and
dark, flre-cured c=bac==s. The cgsul~s obtained ac 5 ~ enzyme/~-m tobacco
are presented in TaDle 2. F=om ~his table £t will be seen Uhau =hls
concentration of enzT=.e releases Lit=Is or no protein from the hurley and
the da=k ua~ac=os. On :he other hand, flue-cured tocac=o showed protein
removal of ac=uu 50% u~der the condi=ions of the experiment.
TABLE 3
ENZYMATIC SOLU3IL[ZAT~ON OP PROTEI~ ZN D%FF~R~~rT TOBACCO TY~
, , , ,,
TOBAC'.O TYPE AND PROTEIN CONTENTT -
HG GE~ DWB
TREATMENT T~ME - HOURS
CONTROL
"NZy,~LE-TRZATED
FL~-~-CUR5~
2
4
6
2
4
6
DARK. .---_UE-C~D
2
4
6
2
4
6
85.0
99.5
89.8
158.0
165.3
167.0
231.3
227.3
234.0
210.5
204.0
192.8
46.8 (45)
45.8 (54)
44.0 (51)
162.S
154.8 (6)
151.8 (3)
229.0
221.5 (3)
229.3 (2)
190.8 (9)
201,0 (1)
IS4,3 (4)
t IgG as s~andard.
The =obaccos were ~=sa=ed with A. orvzae p=o~ease (Sig=a Type XX~:I)
~m'a to~ac¢~) f:~ up Uo ~ hou:s in an environmental shaker at SOeC.
The numbers in b~acko~e are percen= reduc=ions.
(5 mg
B.A.T. CONFIDENTIAL - CATEGORY I: ~|INNESOTA TOBACCO L[TIGATION
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When the ~unga! ~r=~ease :cncsn~rauion was increased to i0 ~g and 20 mg/gm
~obacco, up uo 7C% of ~hs ~r==sin in =he flue-cured tobacco was
solubilized. Zn contrast, only insignlficanu amounts of protein were
solubillzed from the dark, air-cured and dark, flre-cured tobaccos. A
liB:Is more success was c==ai~ed with hurley ~obacco, with about 20% of
~he pro:sin being ~emoved a= 20 mg pr=uease/gm tobacco. These results are
presented in Table 4.
TA~L~ 4
ENZYMATIC SOLUBILZZkT~ON OF PROTEIN ~ DIFFERENT TOBACCO TYPE~
9ROTEZ~ CONTENT -
MG/GM
DWBt
TOBACCO TYPE &
TREATMENT TIME
(HOURS)
~UE-C~R~D
2
4
6
DARK. AIR-CUB~p
2
4
6
DARK, FTRE-CURED
2
4
6
2
4
6
CC:;TROL
IgG as suandard.
T~.O
7£.25
74.00
147.5
13~.5
1;~.0
194.5
i$~.75
166.25
165.0
15~.S
*ENZYME TREAT,~LENT AT
I0 MG G~~ 20 MG G~*
25.S (66)
25.75 (66)
24.0 (68)
131.5 (7)
134.25 (5)
133.5 (S)
196.0
193.0
185.0
159.75 (4)
145.5 (12)
25.0 (67)
23.25 (70)
20.5 (72)
126.75 (i0)
130.0 (8)
127.5 (10)
184.0 (3)
185.5 (5)
184.0 (3}
139.0 (16)
134.5 (18)
141.25 (11) 129.25 (19)
-Prouease (Sigma Type XX;;Z) t:satmsn= a¢ I0 mg & 20 mg/gm ~obacco for up
~o 6 hours in an environmental shaker at 40"C.
The numbers in brackets are per cen~ rsduc¢ions.
~. Hvdrooen Peroxide Treatment of T~bacc¢ U~der Solid-State Condi~icn~:
In earlism s~udies we showed cha~ reduc:ione in Brotein up to 20% were
obtained when cut, flus-cu=ed tobacco (CLX-87) was ~reaued winh 3-6 ~unole
H:O: per gm tobacco a~ 50% ¢oisuure for !S hours au room ~emperature.
La~er, metal ions such as c=p~s~ and ir=n were added a¢ a concen¢=ation of
IO ~mole metal par gm tobacco. However, a~ these concentrations of ms~al
ions rapid heating and char:in~ of the f!ue-ou=ed tobacco occurred, with
copper ~ringing abcu~ a fasts: reaction than iron.
j~
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B.A.T. CONFIDENTIAL - CATEGOP~Y I: MINNESOTA TOBACCO LITIGATION
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Recen~l~, we carried out these reactions a~ lows= concentrations of iron
and c=~per and were successful in =smovLn~ up no 40~ of ~he prone£n from
flue-cured tobacco (see Table 5). We ace ~:w ~epea~ing chase experiments
an ~C~ and 70% ~ois~ure levels. These studies may also prove valuable in
~mpccving the appearance and keeping ~:ali:ies of poe= quality nobacco.
TABLE
EFFECT OF OIIDR.TIVE TREA~'~ENT ON ~ROTEIN CONTENT OF
VTRGINIA TOBACCO UNDER SOLID-STATE CONDITIONS
WEZGHT OF
TREA .-.~.E~NT
WATER - CONTROL
H:O:
Cu: -
Cu: - H.~%.
Fe-'* (i ~mole)
Fe"~ - H~O:
¥e:+ (.= creoles)
Fe-~' - H:O:
TOBACCO RES IDUE
36.0
34.0
36.5
37.5
39.5
37.0
41.0
34.5
P.~OTEIN CONTENT
xsl~x
TOBAC~3 RESIDUE
TOTAL - MG
93.3
63.3 (32)
86.2 (8)
55.2 (40)
81.3 (13)
59.3 (36}
81.2 (13)
67.3 (28)
3,359
2,16S (35)
3,146 (6)
2,108 (37}
3,200 (5)
2,213 {34)
3,329
2,312 (31)
Cu~, flue-cured nobacco was treated w£th H~ or H~ (I0 mmole/gm ~ob.) or
Cu:* (I ~mole/gm sob.) or Fe:" (i or 5 ~mole/~-m no~) or Cu:" - H:O: or
Fe:° - H~ au 50~ moisture con~ent for 24 hours at RT° The tobacco was
~hon cashed wlnh 2 changes of ho~ water, d=igd and p=onein denarmined.
4. Effec= of Medium ComDosi~icn on Pr==eo!v=i= Enzo~e Seqre~an by Yea~q
A large number of yeas~s have been isolated ~urlng a survey of nhe
mic.-o~ial flora of nhe growing ~obac¢o plan: as well as from fermenn£ng
flue-cured nobacco. Many of ~hese yeas=s we=e also screened for
pro~ecl.v~ic acuivity on accoun= of our inte=esn in removing protein from
nobac:: by mic=oDiological procedures. Hewers=0 our inte~es~ resides
mainly in ~hose yeaens ~haU are a~le ~o secrete excess pronease(s) in
liqui~ media, si~=~ it is ~hese ¢ul~uras ~ha: would be useful for ~he
solubilization of pronein f=cm nobaccc.
In a Eesnri=ted screening for enzyme sac=scion, we have Ldennified two
Ha~se~u~a a~mala snrains and an unidenuifie~ yeast aurain, as possessing
~he ahiliny no secrete an acid protease in a simple, chemically-defined
medium. We also have in cur possession a cu~uure of
c~se~:!v~ica~ kindly provided b7 Dr. M.A. i~¢han=e of the ~nive=sity of
Wesne=~ On=erie, London, a~d which has been =epor=ed no he highly
proteci~uic. However, ~his 7easn does not sac=sUe prouease in ~he above
medium. We have now rscogni:ed nhau nhis yeast has ~o he provided a
comple× medium c=nuaining ~=yp~one, which i~ a panc=ea=Lc digesu of
easel=, ~ef=Ee i: will sac=sue an alkali~s ~::teasa in a liquid medium.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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Also, monosac:harides, such as ~i;=ose a~d f=uc::se, when included in ~he
g=o~=h medlu~, shu= down enzyme sac=eti:~ by ~his yeast. Reporued herein
a:e ou~ s~uCLes on the physiological faccocs affec:i~g p:o:ease secretion
~y these ye~5:s.
P~cteclytic activity in Hanse~u!a ~ and cube= ye~s=s was evaluaued
using bovine serum albumin (BSA) and casein as ~ducer3 of p=oteasee.
Since these p=oteLns were incocpc=~ted ~n a simple mod-ium ccnuaining
glucose and ~east Niu=ogen Base (~o la~..i~a acids and (:~H,):S~,) (YNB), six
of cur yeas:s degraded both protslns, hun only H. anc~a~a and isoLaue 20a-
4 exc=eued up to 0.7 unit prouease/ml, cul~ure su~e=na:an~ within 2 days
of gr=wuh. Also, casein appeared uo be superior uo BSA as inducer of
p=o=ease by obese yeasus. C. caseLnolvu~ca deg=aded casei~ bu~ no~ BSA,
and failed :~ excreue protease in :his simple medium.
Since the hl;hly protaolyti= yeas:, C. caseino~vclca failed to produce
pc=tease in uhe simple medium used earils=, we decided ~o tssu enzyme
pc=duo:ion in a complex medium c===aini~; ¢.-yptc~e. Glucose was also
e!imi~aued f=:m ~he medium sLncs iu coui~ suppress enzi~e see=erich.
EazLie= studies had shown that whs~ ~his yeas: is gEow~ on a complex
medium such as casein age= (t:ypuc=e i=cluded), L: produces wide zones of
casei~ hydrolysis. T~us, Gn a t=ypucne-conuaini~g medium :his yeas: now
p=odu¢ed signlfican~ amoun=s of an alkaline pro:ease (0.3~ unit mL").
The
o:he~ yeasua when ~esued on this ~sdiu~, while showing p=ouein
decomposition, did no~ produce exccel!uls: pretense.
i: appears uhau R. one,ale does n=u pz¢duce exoceilula= p=cuease in a
medium conualning uryptone, while C. c~s~o~,~c~c~ needs :he lau~er for
enzyme seems:ion. To confirm these conclusions we decided ~o ~esu for
exoce!lulaz enzyme p~oducuion in ~ diffs=ent media described in Table 6.
F=om :his ~a~is i~ will be seen ~hat C. caseino~v:~c~ produces de~ecuabls
~oun=s of p==usase in medium 1 which c=nuains t=ypuonm and as additional
carbon sour=s, soluble snatch and g~ycerc!, Lnsused of glucose. This
ysas~ fails t= produce exocelluLa: pro:ease in medium Z, which is
dlffs=enu f=cm medium 1 only in ~hat glucose is used insuead of soluble
suazch and glycerol. It also failed ~o see=sue pc=:ease in nhe o~her owe
media which a=s devoid of ~rypuone and c==uain glucose. H. anomala and
isola=e 20a-¢ produce significanu amoun=s of pro~aase in medium 3, bum
none in media cos. i and 2, which connain ==yp=one. Addi=icn of 0.2%
yeast ext=ac= ~o medium 3 (medium 4) no= only slows p==uease production by
=hess :we yeasts, bun also reduces the ~=unts cf enzi.~es uluima~ely
p=cduced.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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H
TABLE 6
MEDIUM COMPOSZT~O~ AN~ SECRETION OF PROTEASE BY Y~.,AST
SUPE.~ATANT PROTE~?Z - MG ML"l
(SUPE.~IATAt~T PROTZASE - UNITS HI."~)
YEAST
H. Anomala
20a-4
C. Caseinolytica
H. Anomala
20a-4
C. Caseinolytica
H. Anomala
20a-4
C. Casetnolytica
H. Anumala
20a-4
C. Caseinolyuica
Z:ICUBATZON PER~OD
0 1 2 4 7
0.90
0.90
0.36
0.90
0.92
0.92
0.94
0.94
0.90
"..00
0.96
0.9a
0.94 (Nil)
0.94 (Nil)
0.ll {0.09)
0.07 (0.04)
0.07 (0.06)
0.94 (Nil)
0.78 (Nil)
o.a2 (Nil)
0.09 (0.07)
0.17 (Nil)
O.IS (Nil)
0.84 (Nil)
0.02 (?;il)
0.03 (?Ill)
0.09 (~.09)
0.11 (:I£1)
0.07 ( :|i!}
0.54 ('zil)
0.03 (Nil)
0.02 (Nil)
0.08 (0.09)
0.06 (Nil)
0.03 (Nil)
O.S6 (Nil)
Nil (0.37)
Nil (0.44)
0.06 (Nil)
Nil (Nil)
Nil (0.07)
0.!S (Nil)
Nil (0.53)
Ni! (0.52)
0.03 (Nil)
Nil (0.18)
Nil (0.12}
0.12 (Nil)
t~il (C.S2)
Nil {C.46)
0.03 (?~il)
Ni! (~.30)
Nil (0.!9)
0.08 (Nil)
Nil (0.51)
Nil (0.51)
0.02 (Nil)
Nil (0.34)
NIL (0.28)
0.04 (Nil)
Msdaum !: YN3 0.17%; Sol. Suarcn 1.0%; Glycerol 3.0%; Tryp=one 0*~%; Casein 0.2%.
" 2: Medium i; Glucose 2.0% instead of Sol. S~arch and Glycer¢i.
" 3: YNB 0.17%; Glucose 2.0%; (NH,):SO. 0.025%; Casein 0.2%.
" 4: Medium 3 plus yeas= ex=rac= 0.2%.
An au=emp~ was made ~o increase p=o~ease secretion by yeas=s by modifying
~he culture media being used. T=yp=one and casein ¢oncnn~rauions were
increased ~o 0.75% and 2.0% respectively, and sucrose was used insuead of
glucose, C. caseino~vnica produced large amounUs of proueasa in ~he media
which conuain 0.75% :=yp=one and casein. Sucrose, unlike glucmse, did no=
suppress enzyme secretion by =his yeas=. ProUease producuion by ~his
yeas= was negligible in ~he simple medium and only a re!auively small
amounu of enzyme was de=eccad on day 7.
In ~he nex~ experimen= ~/p=one concentration was increased ~c 2.0%
(medium no. 2) and sucoose and glucose were compared for their effecu on
enzyme secretion by C. caseinolvtica. From the results of ~his
experimenu, presenusd in Table 7, it will be seen ~haU C. caseino!v~ica
elaborates large amounus of pro~ease in ~he two complex media containing
glycerol soluble e~arch or sucrose as addiuional carbon sources.
Zncreasing ~he nrypnone concenura~ion from 0.75% no 2.0% nearly doubles
~he secretion of procease (day 4 - medium 2 versus medium i). When
glucose is used as addi¢ionai carbon source, pcotease pr=duculon by C.
case£nolvtica is shu¢ down completely and prouein in ~he medium remains
unused even after I0 days (medium 3). Sucrose, on ~he ocher hand, permi:s
procsasa secreuion ccmparable ~o glycerol-scorch (medium 4). Zn a furuher
anuempu ~o increase prouease secretion by C. caseino~vti:~, ~.D% ~Eyp~one
and 3.0% sucrose were included in nhe producuion medium, whe~ 19 unius cf
pro~ease were secrsued on day 2 Isee Table 8, medium 3). This is ~he
highesu activity we have recorded since we were firs~ able ~= de~ec~
exoce!lular pronease secreuicn by ~hls ~ species, and :epresenus
abou: a SS-fold increase in enzyme produculon.
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cAa~z
Zrr-_c¢ oF H~nztm co~POSTT~O, oN PROZZZz~ ozcom=os:.'zo, sY YZ~STq
MEDIUM
NO.
I
3
4
YE~T
C. C~:-':oiy6~
H. An:.-..zli (0-0)
H. An:.-...~z (44-0)
S. Ros='.;s (~'xp llb-10~
Cos:.':[
C. CAs: L-.oiycim~
H. Anc..-.=L~ (0-0)
H. ,~.:.-',b (4.=-0)
S. Ros¢~= (~xp lib-lO}
Cot.u-: i
CULTURE $UPERNATANTS ,':~--STED FOR
PROTEIN CONTENT- MG ML" (PROTEASE ACTrVITY - UNUrS- ML')
INCUBATION PERIOD - DAYS
0 2 4 $ l0
1~
9.1:
11.63
I 1.38
11.38
II.00
8.80
13.00
IZ.40
12.80
12.20
0.12 (3.6l)
6.88
I 1.38
11.38
| 1.00
0.12 ($.78)
I0.7~
II.13
II.~_5
I 1.35
C. C.ls e..-.~i~ca
H. A~.o-~L~ ((NO)
H. Ano.m:zt~ (at-0)
S. Rosc:.:s (l:xp 1 lb-lO)
Con:.-.i
C. C~s,---oiyzic=
H. Anc-..~b. (0-0)
H. An.c-.~L~ (4.4-.0)
S. Rose,s ('Pxp llb-10)
Con::'d
~odLum i:
11.25 10.25
11.~-5 10.63
11."-5 11.00
10.88 0.$8 (0.01)
iI.~3 iI.25
7.13 0. I1 {4.00)
10.88 9.63
10.g8 10.88
10.75 0.gg (Ni/)
10.88 I I.P.~
0.08 (6J5)
0.70
11.38
1.30
11.00
0.10 (I'~.14)
7.63
9.:5
II.75
i 1.88
I0.00
7.00
I0.$0
1.30 (Nil]
II.00
o.~o (6.68)
$,63
10.63
0.51 (Nil)
11.75
0.0": (5.87)
0.': (Nil)
1.0.c (Nil)
0.:~ (NiJ)
I 1.38
O.OT 14.09)
3.?o (Nil)
Z..~0 (Nil)
1 I.SS
12.00
ii.00
:.35 (0.01)
8.T.c (Nil)
0.-~-~ (Nil)
0.0S (0.$8)
3.~-Z (Nil)
7.13 ,.'0.~)
0J0 (Nil)
11.25
0.~
1.60
0.T$
II.8S
0.09 (.~.32)
3.$0
1.90
IZ.O0
iZ..~O
I0..~0
~.7~
0..~
~I.~8
0.09 ','0.39)
~.00
6.~
0.10
I'(30
4:
0.17 (0.30)
12.00
0.Zl (0.3Z}
12.Y.~
O.O9 (0.09)
II.S8
YNB 0.17%; SoZ. Starch 1.0%; G~yceroL 3.0%; Try~Cone 0.75k;
CaseLn 2.0%.
Medium I, excep~ Tryptone 2.0%
" I, Glucose 3.0% instead of Sol. Starch & Glycerol
" 1, Suc=ose 3.0% .......
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MEDIUM
NO.
T~LE 8
PROTEASE PRODUCTION BY CANDIDA CASEZNOL~!'.$CA
C~ITURE SUPERNATANTS TESTED FOR ~ROTEIN COW,TENT - MG H.L"
(PROTEASE ACTIVITY - UNITS ML"I-
C S HR.
I0.~ 9.~0
C0.CZ) (0.06)
i0.6~ 4.7~
(0.~) (0.12)
10.~ 4.2~
(0.i:) (0.17)
INCUBAT'.ON PERIC." - DAYS
2 4 I 5 j 6 7 i 8
0.46
(0.599
0.29
(0.66)
0.18
(1.0~)
0.12
(3.84~
0.14
(i0.3~
0.2C
(!9.12)
0.i0
(7.77)
0.i!
(7.71)
0.17
(4.86;
0.10
(7.10)
0.I0
(6.23)
0.18
(1.43)
0.09
(8.~2)
0.09
(4.86)
0.1a
(0.49)
0.09
(9.45)
0.08
(S.63)
0.18
(O.S2)
0.i0
(5.42)
Medi'~ I: YN3 0.!7%; Sol. 3ua=ch 1.0%, Glycerol 3.0%; T~/p~one 0.7~%;
Casein 2.0%.
2: Medium I, excapu Tryp==ne 2.0%
3: " 2, S~crose 3% insuead of S:l. S~arch and Glycerol.
*HPA as subsuraue, i uni= = ~ 10.D. MI~'M~ ~eac~ion mixture a~ 37eC
and cH 9.0.
Since in¢rsasing ¢.-yp~cne and casein concentrations in ~he complex medium
used for C. caseino!v~c~ gave la=;e increases in prouease production by
~his yeasu, i: was decided ~o a~uemp~ to increase prousase production by
H. ancmala culuures by increasing ¢asein ¢c~:sn~raUion in uhe simple
medi'=m used f:= enzyme production by ~ese yeasts. S~crose and glucose
were also ccm;ared for :heir effec: on prouease produc=ion. The resul~s
of =his e~p.e:imenu wars a ¢ompleus surprise. The ~wo sur~ins of H.
failed ~o secrete pro~easa in naese =edia, even nhough ~hey are
known ~o do so in similar media. The only difference here was ~hau ~he
concanurauion of casein was increased ~o 2.~ and 5.0%. The results show
~hau such high concen~rauions cf casein suppress pro~ease secretion by .=..
anc~..~ia. C. caseino~Vuica, too, did sou decompose casein in ~hese simple
medla, includ&ng medium 2 concaining sucrose as addi¢i=nal carbon source.
Tha= =he high casein ccncenuraUicn suppressed prouease secretion hy8.
was ~ickly confirmed in :he sex= experiment in which casein was
inccrpora=ed au 0.2% and 2.0% concentrations. In ~his experimen~
Har~ersUein grade casein was compared wish sodium caseinaue, hcuh supplied
by I~. Both cul~ures of H. ancma!a ~es~ed, secreted significan~ amcun=s
of prDuease in the uwo media conuaining 0.2% casein and, a~ mos~, ~Eaces
of pr=¢ease in the nwo media conuaining 2.0% casein. The type of case/=
used did no~ appear Uo significanu!y influence pro~ease secretion by ~hese
yeasus. A culzure of Sz:~obo~omvz~s roseus examined in uhis experimenu
turnel out to be a poor producer cf pro~ease.
In conclusion, i~ should be poinuel DUO ~ha= we are n~w beginning ~o
idenzlfy uhe c~ndiuions under which ~hese yeasts will secreue opuimal
amounts of prczeasas in ¢he ¢ul~ure medium. Only when we fully learn ~c
conzrcl and c~=imi=e pr:=~ase production by ¢hsse yeasus, will be suc:ea~
in cur uluimaue goal of propels removal from =obacco by yeasu ~rea~men~.
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3. P~oteLn Con:en: of Ci~e~:e S~oke C~densaue
Earlier protein de~e.~u, ina:i:ns in cL;areuUe smoke ¢ondensa=es showed
unreal£suic amoun=s of pEous£n suqges~ing inuerference by phenolic
substances in the pEouein ~s~e.-m. inauion procedure. S=¢h an Interference
Ls to be expected in an exu:e¢e~y l:w prouein material such as ciga:euue
smoke ¢ondensaue. In the ~:ess~u scud7, phenolic mauerial was ex~:acued
prior ~o prouein deue~minauion ~y uhe Bradford procedure. The prouein
con~en~ of condensaues fr:= 2 cigareuues, afuer repeated euhanolic
exuEacuions, is pEssenued in Table %. Yrom uhe ~a~e i~ will be seen ~hau
after 2 e~hanolio sxu:acuL:ns no fur:her removal of phenolic manet!el
occurs. Of course, i: is possible uhau large pepuide fragmenus, soluble
An e~hano!, are losn in ~h~ repea~ed exuracnian. Acuua! protein Iso!a~ion
from condensaue could ~esc!ve uhis p:cb!am.
TABLE 9
PROTE!NCONTE~ OF CIGARETTE SMOKE CO~DENSAT~S
SMOKE CONDENSATE
FROM
PLAYER'S CK 28
GAULOISES
PRO~E:N CONTENT - ~ AFTER
0.7.5
0.2.2
0.24
0..~3
0..~.=
0.35
i 2 3
ETY~OL EXTRACTIONS
0.13
0.13
0.13
0.16
0.20
0.18
0.13
0.12
0.13
0.22
0.18
0.18
Phenolic material was extracted frc~ ~he oondensates with s~hanoZ. The
pro~mins were pelleued by =entrifugauion and redissolved in 0.i M NaOH.
Protein ¢:nnenu was de~e~..i~ed using the Bradford :eagenu.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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BATCo document for PFSFC 1 March 1999
o.
RECORO TYPE:
SU@ TYPE:
SECURITY CODE:
FUNDI~;G BODY:
ORGA~;~ZATION:
GROUP NUHSER:
LOCAL PROJECT NUH@ER(S):
PROJECT TITLE:
PERSQ~ RESPONSTSLE:
EFFORT:
PROJECT DESCRIPTIOfT:
SCOPE:
DEPTH:
FUNCTION:
OBJECTIVE:
CLUSTER:
DATE..REVIEW WRITTE:;:
REVIEW TIT',E:
IT1 CA::A~A
41~
T-CI11
Non Rouulne A-~aly~ical ServLce ~o Suppo~
R ~ ~ Projecus and for General T=cub~eshoo~ing
POUIIN, P.; DUMONT, J.
!9~
Work under uhis projec~ is designed ~o provide
Anai~ulcal Su~o=~ for ceques~s made by
Pur=hasimg, TJchnical Secvices, Manufacturing,
Ma:keui~ and R & D. In addiuion any
mon~u3clng ~o satisfy goveEnmen~ guidelines
faii~ wiuhin uhe scope of ~his pc=je¢=.
LOC~
SUPPORT
SU~:Z~T:VE
ME~E~D DZV
Jui-! 1994
Non-Rouulne A~a!y~ical Projects ~o Sup~oE~ R&D
and • General Troubleshooting.
Pr::s~ s .=r
REVIEW TEXT: 5in:s uhe lasu review (JanuaF~ 1994) ~he
following se=vLces have been cender~d:
I. Scearm. in~ Ana!vs~s o~ Ca~o Sc~:~a~:
Cameo Special will nou be pEodu:~d any more.
2. Oual£~v ConC~=! of MenThol C~vs:~!s for Gu~Dh P~an~:
SixTy-Two (62) random s~ples of menuhol ::ys~als from 2 ehlpmen~s have
been analyzed since January 19~, amd fou~ ~o be within specifications.
3. Mo~L~o~in~ of Humec~an~ Levels !~ IT~ and OoDosi~on @~andq:
Glyce~ol levels we=e checked f:r .~R-Mac~:nald and RBH pr=duc~.s. On a
bone dry-weigh~ basis :he glyce=:! levels for 1994 were:
C~ave~ "A"
Hark Ten
Expo~ A
Expor~ A Lighu
Expo~u. A UlUra Limb:
Exporu A S?BL (11C g)
Export A Lighu SP~I (113 g)
Relvedece S~pe~o!i 200 (135 9)
Selvedere SUP.20G :: (90 g)
GLYCEROL
ls~ 2nd
QTR. QTR.
3.65 2.78
3.26 2.50
4.46 4.68
4.73 4.32
4.31 3.99
3.14 3.60
3.97 3.61
3.90 2.55
4.22 2.98
T= suppor~ d£ffe~enu pr:~ecus (T-3~1~, T-3:~4 and T-4920) ~we!vs (12)
samples, of American ci-~areuuss _n.= ~ cuu ~=~::0 have been analv=ed.
~o
deuermi~e gIyce:~l and ~r=py~en~ ~I~:=i !s~els.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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To suppc:= =he Hiddls-Eas= prc~ec= (T-3224) ~irCy-cwo {22) cigarecue
samples have ~een a=alyzed far glycer:i and propylene gi~:=l levels.
To suppc:= pr=~ec= T-5084 fi~=y-elgh= {Sai sa~les og :-~3 ==bacoo from
Corby were ues:ad f=r glycerol leve~s. Glycerol were measured Ln samples
before and af=er expansion Uo evaluaue =he glycerol losses during
expansion. The ave:age glycerol loss during expansion ;ere;
D-6C: 8.!% : S.7% (n - 19)*
* Average resul=s fc= 19 deuerminau!:~s f::m December !~i ~o May 1994.
4. Consume~ Ccm=!a~n~s ~ves~iaa~ans:
Since January 1994, specific analyses ~ere =equired for five (5) consumer
complain=3 (s~e ra=e as las~ year).
co,scaR COMPLA~TS - :~NUARY TO ~ ~99~
RESULTS
DESCR:~T~O~
SUBJEcT:YE
Oily substance
Oil spocs/3=own spots
T=bacco like glue/
Znsec~e?
T:bacco locking bad/
consumer sick
Consumer slck
Nag.
~:~ALYS IS
Mine:el o£1.
?araffL~ oiZ from
alumi~''~ foil/~cbacco
produc=~.
Moulds:':~o insects.
Moulds and bao=eria.
Old o= ¢verdriid
~obac=:.
No,-Routine Ex=~qEau=~v Work f~r Producu Oevelocmen~ or for General
T=oublssh:c=inc Ln Plants:
In January, plasticizer levels have been deuermined £n f=~: (4} samples of
fil~er rods ~o suppcr~ a production uisu. Analysis was hy ga~
chromatography using methanol as ~he ex=racuing solution, chio=ina~ed
solvents (Yreon 113 cr mechylene chlo:ide) are no longer :sad for ~his
procedure.
In March, a black fo~ rubber (insulaulng rubber) from =he RBH Dieu plan~
in Brampucn was analyzed ~o deuermine !us composition and =o assess ~he
impac= of accidence! blending w£~h ~c~acco. The manerial ~as found ~o be
an elaen:mer meaning a vulcanized syn=henlc ru~ber. Z~ ::n=ains polyvinyl
chloride as a flame reuardann, chlori~ausd paraffins, a!k~iphUhala~e,
plasuicize:s, s=abi!i:ers, eUc. This =auerlal should non ~e used in a
manner such ~ha: risk of contaminating smoklng ~obacco p::duc~s exisCs.
~n March, =he compose:ion of oily spcus found on cigareuuss made ~iCh
MaUin~e Fine Co= (5C ; pouches) was dece=m.i=ed. The oil ~as iden=ified as
Esso P=i~..cl 3S5 which is a mineral o;i havi~; no affec= c~ che ¢as=e of
smoking ¢=~accc prcdu=us. The source ~as a leak on ~he $¢P~ermunds
packing =~chine. A~:upriaUe ~c~ific~=icns have been =a~e :n ~his
=achine.
B.A.T. CONFIDENTIAL - CATEGORY I: ~'~INNESOTA TOBACCO LITIGATION
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Zn April, analyses were performed to determine ~he ~:opylene ~l¥=ol
peneura~ion rate in frozen and unfrozen tobacco stems. ~urin~ winter,
Montreal planu receive stem at a temperature below 0=C. The boxes are
s~ored unuil the stmm reach room ~e=perature. The analysis revealed no
signifiuanu differen=e in the propy!ene glycol paneurauic~ race for frozen
stem oomparQd ~o uhawad stem. The storage ~ime used to uhaw uho etlms can
be eliminaued.
B.A.T. CONFIDENTIAL - CATEGORY I: M[NNESOTA TOBACCO LIIqGATION
¢D
~r-
rxO
L2~
£24
BATCo document for PFSFC 1 March 1999
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"~" CA::ADA
T-7282
Reduction cf Irritation
PORTER, A.; McBRiDE, C.
0.35 - 1994
A range of tobacco, paper and fi!~er addlUlves
will be screened ~o assess :heir effect on
reducing smoke irritation. The additives will
also be eva!uaued foe possih~ie negative changes
in smoke quality, s~ability, cost and ease cf
application.
GROUP RELEVANT
FUNDAM.E~TAL
GENERAL
ALTE.~NATIVE
PROD~CT RES/~EV.
$uly i~94
Reduczlon of Irritation
As described in the lasu review, addition of
~:A~ON CR-20 resin (either 50 or I00 mg) ~o the fil~ers of du Maurisr K.S. was
successful in reducing aldehyde levels by ~0-80% with concomitant dec=eases in
perceived i=ri~a~ion. Eased on ~his, 2 other ion exchange resins, AT~erllue
:.RA-93 and LA-2 were ~ss~ed. These were evaluated using 2~ and 50 mg resin
added to ~he fil~e=. Another evaluation of CR-2Q was also perf=rmed a= 25 and
~3 mg.
The A~er!i=e IRA-93, even after freeze-drying, had an exCreme!y scr:ng fishy
cdour. Incorporation of the resin into a gap configured fi!~er masked any
overt off-odour hu~ ad-hoc subjecuive eva!uauion showed enough transfer ~o ~he
=ainsuream smoke ~o warran~ a hal~ in ~ssulng ~hie resin.
The LA-2 ion exchange is in li~aid form. To inc==pora~e ~is ¢n=c fil=eEs ~o=
evaluation, paper filter rods were soaked in methanol eolu=icns of LA-2 of ~he
appropriate concen~Ea:ions to :esul~ in 3 levels of LA-2 pe: I0 mm fi!ue=
segmen=: 5, IS and 25 mg. Soaking the fil~ers in ~his fashi=n caused a
Eedu¢=ion in pressure drop (approximately 3%). However, ~he paper falters did
nc~ a~sor~ the resin comple~e!y and over a few days the resin began uo desorb.
T~is, uogeuher wi~h a re=her s:rcng odou=, hal:ed ~his line of inves:iga~icn.
Ce~lu!~se aceuaue filter rods were deformed after soaking in LA-2 anl could
noK be used.
Tas~ing continued wi~h DIAION OR-20 added to fil~srs a~ 25 and 50 mg weights.
• reeze-dried CR-20 was used and in addition 2 levels of char=tel, by weight,
were added ~o minimize any possible ~as~e modificauions. The weights of
charcoal added were 5 and 10% of ~he weight cf CR-20 resin (2.S and S.~ mg for
2~ mg CR-20 and 5.0 and i0.0 mg for 50 mg cf CR-20]. These oroDoruions were
considered sufficiently small n=: ~o impart a charcoal off-~asua. Ad hoc
sub~ecUlve evaluation indicated no~iceable reductions in irritation with no
apparent ~asue quality effect. More samples in this series have been made and
wi!L be eva!ua~ed by ~he Expert Panel.
An=ther line of inves=iga~ion has been initia~ed in order ~3 assess =he
contribution of vapour phase cc=ponenus ~o smoke irri~ation. The use of
danuder tu~es to collect aldehyies and vapcur phase nicotine has been t~sued
using c3nventiona! cigare~=es. ~nitial studies have shown ~ha~ the :=mbus~ion
cf clgareu:as with denuder tubes in place has a small effezu cn mainsu:e~ T~M
deliverles (5% decrease).
B.A.T. CONFIDENTIAL - CATEGORY I: ~V[FF/NESOTA TOBACCO LITIGATION
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Opuimi:a=L:n a=. ~e meuhodoloqy, se!l¢=~ve
¢oa~'~-~s ~o ¢o;!ec= specific vapou:
phase c::~oun~s an~ GC and HPLC analysis wLll be undeE~aken By a Co-op s~udenu
£n Sepue~=er.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
O
(..F~
BATCo document for PFSFC 1 March 1999
- 21 -
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ITL CANADA
416
T-9642
Developmenu Of a Procedure for ~he
OeueE=ina~ion of Possible C=nuaminanus in
To~acc: and Cigareuue Samples
9OULIN, P.; DUMONT, J.
1994
To develop as ex~ensive as poss~bls ana!~ical
procedure for ~he analysis of ~l~egsdly
connaminaned samples. This will ensure a
ber~mr evaluation of cQnsumeE compLa£n~s and
will assisu Manufacuurlng when an Invesulga~ion
of an oil ~r greass leak Ls rs.~T~i=ed.
LOCAL
SUPPORT
GENERAL
SUB~EC'. IVE
M~THOD DEV.
July 1994
Developmenu of a procedure for ~he
deuermina~£o~ of possible contaminants in .
uobac=o and =igare~:e samples.
The existing da~a base (chemical and physical
c~aracueris~Lcs, ZR and UV spsc-.Ea and ohroma~ograms) of the oils, greases and
so~ve~:s used in ~he planus are being upgraded foe any any new lubrican=s.
This '~rk Ls an ongoing pr~eo=.
An L~-_r:vsd GO/MS me-~.od foe ~he de:ec~ion of ~races Of ~il t~ assess ~obaco:
conuaminaUion is in ~.evelopmen:. This meuhod was fi-~su developed by Rou.~=an's
Tnne.~.--...~ional and ha~ • purposed ~e~e=~ion ~evel of be~e= ~han 0.01%. This
E
msuhod uses Uriu•~'.ga-ss and sue anes as ~a=ksrs for fossil fuel oi~s. At
a •
posses-- experiments .- bel.ng performed on pure oil. However, sensiblY!'-7
p~ob~e.~.s have been e.~rounue.-ed.
B.A.T. CONFIDENTIAL - CATEGORY I: ~IINNESOTA TOBACCO LITIGATION
t.."
t..F
C',
BATCo document for PFSFC 1 March 1999
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OS.'ECTIVE-:
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DATE REVIEW WRITTEN:
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REVIEW TEXT:
Methods used in ~ & D:
~TL CANAZA
415
T-~549
Review an~ Development of Altarnative
Analy~iczl Me=hods
POUL~N, P.; DUMONT, J.
1994
The ori~izal objective was ~o develop
chromau=~raphic me,hods re!cued to ~he nature
of the c=~pound uo be analyzed (GC for volatile
components, HPLC for non-volatile). The
oujscuive has been expanded u= include
analytical methods in general ~O meet ~hs
changing requirements of R & D.
LOCAL
SUPPORT
GE:~ERA/.
SU~JECT:'.-~_
METHOD DEV.
July 19S4
Review an~ development of alternative
analytical methods.
I. Annual Audi~ of Ana!v~ica! Needs ~n~
Gas chromatographic me,hods used for non-routine
analyses are presently being reviewed. Uachni=al improvemen=s such as the use
of Merlin Microseal Sepua instead of re~u!aE sepua and more efficlan~
inJec=ion uu~es are being seu up. All =he ch=oma~ographic conditions such as
flow and pressure of helium and ~smpera=ure are being verified to improve the
chromatography and di~inlsh the analyti:aL time.
2. DeveloDmen~ of a Msth=d for the Anai':~Ls of Free Amino Acids i~ T~ac~n:
We have evalua:ed a new reagent Accq.Tag chemistry package co~ercialized
by waters for the analysis of amino a=ids. The reagen~ is 6-
aminoqulnolyl-N-hydroxysuccln£midyl :arbamate (AQC), and works wi~h
prlmaEy and seconda~I amino acids. The sample deriva~iza:ion proo~uEe is
supplied by Waters, an~ we have verified ~hau i~ can he used Uo analyze
free amino acids in t=~accc.
The ~u~ tobacco s&~ple C2 g) is extracted winh 50 mL of HCl O.l N for 2
hours on a WrisU-Aculcn shaker. The extract is centrifuged au 2500 ~m
for 15 min., and fi!tsred nhrough 0.4~ ~m. The sample is ~hen ready foe
deriva~izaticn.
The individual amino acid levels vat? considerably within the same sample.
The mos~ predominan= ones (glu~ami= acid, his~idine, arginine, alan!he,
praline) are dilutad following ~he ;=~cedu~e described in Table i fc~
sample I-A. For the ~inor ami~o acii~ the derivatives are ~repared
following the procedure for Sample i-~. These ~wo derivative procedures
allow us ~o quantify all amino acids =i~h only one set of s=andards.
B.A.T. CONFIDENTIAL - CATEGORY I: ~IINNESOTA TOBACCO LITIGATION
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rxD
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BATCo document for PFSFC 1 March 1999
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TABLE I
DERIVP~TI:VE ~AMPLE P.".EPARAT~.OHS
Sample exuracu
:nuernal suandaEd
Warier
Toual
r
VOLUP--S (~L) USED FOR
MAJC.~ AHZ:;O ACDDS
SAMPLE I-A
30
32
10C0
VOLUMES (~L) USED FOR
MINOR AMINO ACIDS
S;~HPLE 1-B
968
32
0
1000
Examples cf chromauograms of a suandar~ series of amino acids (40 l~ol/~L
for each except cystlne which is 20 ~=cl/#L) and an exurac= of Canadian
flue-cured :ohacco ace given in Figu:~ I.
~eak iden~ificauion and re~snui=n tL~e are given in Table 2.
~EAK #
TABLE 2
AMINO ACID
NAME
1 Hydmoxyproline
2 Aspa¢'.ic acid
3 Se=ine
4 Olu~amic acid
5 Glycine
Histidine
7 AEginine
8 Threonine
9 Alanine
I0 P=oline
11 a-aminobu~yric acid
12 CysUine
13 Tyrosine
14 Valine
iS Me~hionine
16 Lysine
17 Isoleucine
18 Leucine
19 Phenylalanine
A33REVIAT:ON
HYPRO
ASP
SER
GL~
GLY
RETZNT2ON TIME
(min)
17.93
20.80
22.56
23.66
24.84
HIS
ARG
THR
ALA
PRO
AAbA
CYS
TYR
VAL
M--~T
LYS
ILE
LEU
PHE
25.39
27.16
27.36
28.06
29.21
30.63
31.85
32.71
33.58
34.25
36.23
3~.76
36.54
40.81
The nex~ suep will be to evaluaue ~he reproducibility of che me~hod and
validaue i= by compaEing ou= results wi:h o~her, mo~e well established
~rocedures.
3. :~p¢cved Me:hod fo~ tenzc{a}ovreD~:
A me~hod was developed at ZTL in 1989 (:TL Me¢h¢d #28) tc analyze a(a)P in
~PM. This procedure al~ows ~he de~e:~...~na~ion cf approxi=ausly 2
samgl~s/day.
B.A.T. CONFIDENTIAL - CATEGORY I: ~4[NNESOTA TOBACCO LITIGATION
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This severely li=i=s ~he feasibili=y cf using =~is meuh:: for routine
analys£s.
In method #2S, ~he car is collected on a Cam~rid;e pad a=d B(a)P is
exurac¢ed by shaking in cyclohexane (i mL/l mg WTPM), filtered and
evaporated uo < 10 mL. This filtra¢e Ls diluted to 10 ~ with
cyclohexane, soni¢aued and re-filtered. The s&uple is ¢hen ready U: be
cleaned over an NH: Sep-Pak ca~ridge. After :he Sep-Pak has been
conditioned wi~h hexane, an aliquot, of 1-4 mL 3f the filtrate is added ~o
the cartridge.
The method has been streamlined fu~her using 43 n~L of c-!clohexa~e
regardless of weight of WT~M. With this modifi~atio~ iU is possible Uo
avoid the evaporation s~ep. After the Sep-Pak has been condiuioned, an
aliquot of 4-16 mL of the filtrate is added ~o the ca1--.rldge.
The resul~s obtained by the owe procedures are as foe!owe:
METHOD
1 m~ cyclohexane/1 mg
of WTPM
40 mr. cyclohexane
B(a)? I~ FTC ~AR
(n~/mq)
IR!
4 0.76 = 0.14 0.8.~ -" 0.18
4 0.93 -~ 0. !5
B ( a ) P DELIVERY
IR4F I IRI IR4P
l
26.6 .- 4.2 8.5 -" I.I
0.9-~ -" 0.15 29.2 = 3.9 8.7 -" !.I
tPive cigare=ues were smoked per pad.
According ~o a s~udenu'e t-~es~ (p < 0.05), there are no s~a~istlca!
differences (p • 0.05) among ~he reeu~.us with eluheE procedure.
This method can be used to analyze 4-6 pads/day for benzo(a)pyrene, which
allows ~he method uo be ~sed for routine analyses.
4.
Deve!o~men~ of a Me,hod for ~he Analysis of A1~hydes ~3 ~a~s~eam
Ci~ar~t:e S~oke:
ITL has a procedure ~o measure aldehydes in ci~are~:e suke based on
Houlgate's work (I). CigaEetue smoke is ==llec:ed in Drachsel bo~!es
containing a soluuion of dinit~op~enylhydrazine (DN~H), which reacts wiBh
aldehydes u= form phenylhydra=one derlvauives =f the corresponding
aldehydes. One ma~or disadvantage of this me~h=d is that ~he derivatives
are not s~able and the sample mus= be injected cn ~he H~C within 30 men.
of being collected.
This drawback e~iminates auuomation of the analysis. In o:de~ to avoid
this problem, iU was decided to evaluate the possibility of separating the
collection and derivanizauion into 2 steps. The aldehydes in smoke were
collected in a so!vent (firs~ step). The derivauizauion step was delayed
until just prior to the HPLC analysis us£ng an auuoinjecu=r to perform a
preco!umn derivauizauion of the samples. We used ~he 71~ UIu:~ Wis~
Au~oinjecuor from Waters in ~he AuUotransfer mcde ~o perform the
precc!umns derivauizauion.
We knew that s~andard solutions of aldehydes i~ ~a~er are stable for 7
days (Ref. ZTL method no. 25) (2). For Bhis reason, i~ was decided ~o t~
2 col~ecUi=n solutions: water and water ~ per:nlori¢ aci~ (~he catalyst).
The ~erlvauizanion steps are illusu=a:ed in ~i~ure i. Different
paras.scots were varied uo opulmize the :sacuic~ such as :he nu~er of
mixi~ cycles and =he rear:ion delay. Both opui~izaui:~s are presented in
B.A.T. CONFIDENTIAL - CATEGORY I: ~IINNESOTA TOBACCO LITIGATION
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Figu=es 2 and 3 ~es~ec~ively. :wo mixi=g cycles and a :;=e de~ay of 12
min. were selected. These co:respond := the optimum con:i:ions for
acetaldehyde and ac~ole~. The opuimu= conditions far fc.-m, aldehyde are 9
m~xi=g cycles and a 30 min. c~T~ delay, 3uu ~hese c=ndi=~=ns represent a
ma~o= decrease for ~e a=he= aldehydes. FoP this tease=, £c was decided
co use che optimum condi=ions f=~ acetaldehyde and acro!eln.
To verify the aldehyde suabillutes in different col!ecui=n so~uuions, a
sample and a standard were £n~ecued over a certain peric~ of ulme. The
standard was used ~o eva!cane instrumental drif~ since, as no~ed, ~he
scandards are suable in water f=r 7 days. Therefore any varia¢ion in
standard aldehyde leve~ ks due u3 insU=u=enual drift no= degradations
The sample results for formaldehyde, ac~caldehyd~ and acrclein collected
in wa=er only showed dec:eases ¢ve= IS h3urs of 27, 54 an~ 37%
respecuive!y. The standard results showed decreases of 14, 41 and 24%
~especuive!y. There is instru=enual d:~f~, but ic cannot explain ~he
dec=eases of the aldehydes for the sample, therefore the =igarsc~e smoke
mac=ix is probably affec=ing the suabillc7 of the aldehyc~e.
In the case of the second trapping eo!uui:n con=~ini~g we=st and
perc=!oric acid, ~he form. aldehyde, acetaldehyde and act:lain levels in the
sample decreased by 3, 41 and ~% reepe¢ulvely over a pe=iod of 13 hours.
For :he standard the dec=ease was 4, 2C and 31%. The fc==..aldehyde and
ac=clain decreases can be exp~ai=ed by i=s~=umenual drift hut ~he
acetaldehyde decrease canno~, and we co=eluded chat the lance= was
unsua:le in this collsculon solution.
Since aldehydes are soluble Ln alcohol, a third ~rapping ~uluulon using
methanol was evaluated. In ~hls experiment, ~he sample fc--maldehyde,
acetaldehyde and ac=olsin levels varied ¢ver a period of 15 hours by 2, 8
and 45% respectively. The standard inje==ed over ~he sa~.~ pe~iod of time
showed varlauions of 0, 9 and 3~% respeculvely. I¢ seems :hat variation
for sample aldehydes coLlec=ed in mec~a~cl is due mainly co an
instrumental drift and nou ~o deg~ada~ic~ of ~he aldehydes at leas~ Eor
formaldehyde and acetaldehyde. The inscyumen~ drift cannot explain ~he
yetis=ion foe acro~ein.
However, a 10% degradaci:n is =ela~ive!y low over a I~ hot= period.
These cesu!ts ape preliminary a=d will ~e confirmed, but i: appears ~haU
mul:ipLe smokings and col!sonic= Of aldehydes =o permit ha:oh analysis is
~easi~Le.
(1)
(2)
?seer R. HoucgaCa, Kubhi S. 0hingra, Stephen J. Nash end William H.
Evans, Analyst, Vol. 114, 2~5, 198~.
:TL method no. 26, "High ~e=formancs Liquid Chramauc~=ap~!¢
DeUer~...ina~ion of Aldehydes in ToDac:= Smoke", So:she= 1993.
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R~VZEW T:TL=:-
IT1 CANADA
T-~578
Devslopmenu cf a Protocol ~o Es¢a~!ish Residual
Sclvenu Upper Limius in Packaging MaueEiaL.
D~MONT, J.; DUNN, R.; LETOUR~NEAU, C.
1994
Developmenu of a protocol ~o establish residual
sclvenu upper limius in packaging material.
GROUP
FU:;DAMENTAL
GE:;ERAL
Jui'! 1994
Developmen~ of a pro=cool to ssua~!ish residual
s=ivenu uppe~ limits in packaging malarial.
R~V:EW TEXT: A pro=o¢o~ has been developed uo svalua:e
resldua! solvenus or compounds in packaging material Bha~ may uransfer :o
c~'~a=euues and modify u~e ~asue of uobacco producus.
A subjec=ive evalua=ion pronocol has also been developed, which includes ~he
simple prepara¢ion (exposure mode and ¢ondiUions). A subjecuive evaluaulon
scale (:ndex) has been created wizh a more desoripuive definluion of ~he ~as~a
mmdlfica=ion and is presenued in Table I.
To evaluaue ~he normal {intrinsic) subjective evaluation varlauion, ~he same
sam..p!a was evaluaned over 58 days. The evaiuanion is repor~sd in Table 2.
The sub~ec=!ve index varies between 1.4 and 3.7 for similar residual solven=
levels. However uhe subjective index is lower and somewhaulsss variable
afu~r 30 days of exposure ~han i= is for 1 and 7 days. We will =epean Bhis
~es~ ~o verify ~hese observations.
The nex= suec. will be ",3 invesuigaue possible relaUionships beuween ~he
solvsnu levels and subjective evaiuauion, u3 be able Uo predic= i" packaging
malaria~. can be used or no~.
A reporu will be issued during uhe su~er of 1994.
B.A.T. CONFIDENTIAL - CATEGOP-.Y h ~IINNESOTA TOBACCO LITIGATION
P~J
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- 27 -
SC.'-.I "
0
Z.:
2.0
2.5
3.:
3.5
4.,3
4.5
5.~
6.0
7.0
8.0
i
2
2
TABLE I
DESCRZPTION
No difference.
Marginal differences,
S1i~hu differences.
$1ig~tly modified strengnh.
Slightly modified issue or nouiceabl7 modified s~reng~h.
Slight off-taste on firsu puff then sLightLy ~,odLfie~ ~as~e.
Slight off-taste on first few puffs then no difference.
Notlceably modified basts (noticeable mouthcoauing).
Sli~h~ off-~asue on first f~ puffs uhen modiZied ~asua.
Small off~tasne all cigareuuss.
Off-users all cigaEs~ues (noticeable).
Surong off-~as~e all ciqare~ues (ver~ no~iceah!e).
Averages below 2.0 are considered accepuable.
Averages between 2.0 and 3.0 are moderaue in severity.
Averages above 3.0 are unaccepuable.
TABLE 2
SAMPLE ~ Is?roH
Ref. 11S
Ref, 117
Ref. 120
Ref. 121
Her. 122
Ref. 123
Rsf. 124
22.0
2~.0
22.0
2:3.0
24.2
24.6
"'.0
.g/cig.
EnAc
,,,,
3.1 S.G
,~.2 4.3
2.5 4.2
2.7 4.7
2.9 3.8
2.4 3.6
IsPrAc i n-PrAc
3.4 3.1
3,7 2.5
4.1 2.8
4.5 3.5
4.0 3.1
3.8 2.8
1.5 1.7 2.2
2.0
EXP. T~ME"
(days)
1
7
30
39
45
81
5a
,, ,,,,
SUS~CT, ZVE
INDEX
3.7
3,3
1.6
2.35
1.65
3.0
1.7
1.5
1.4
- Ex=csur~ time of cigare~',es wluh packaging mate.-ial.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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WORK AREA 03
SMOK =.~;G 8E~VZOUR
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RECORD TYPE:
SU~ TYPE:
SECURITY CODE:
FUN~ZNG BODY:
ORGANIZATION:
G~OUP NUMBER:
LOCAL PROJECT NUMBER(S):
PROJECT TITLE:
PERSON RESPO:{S~BLE:
EFFORT:
PROJECT DESCR:~TION:
SCOPE:
DEPTH:
FUNCTION:
OBJeCTiVE:
~USTER:
DATE REVIEW WRITTEN:
R-~VIEWTITLE:
REVIEW TEXT:
I. PRODUCT ET.AST~CZTY:
ITL CANADA
441
T-8079-1;8079-2;8077-17;8077-18;B077-20;
8080-1;8080-3
Smoking aehaviour
YOUSSE~, M.
2 " 1994
The objective of ~he smoki~ behavlour program
is ~o provide a scientific und,=standing of the
inneracuion between the smoker and the product.
To ~his end ~he recording system is being
upgraded for improved ease of use, precision
~= rate at which dana can be generated
(T-8079-I). Changes in smoking behaviour
resulting from produ¢~ n~:difica~ions will be
investigated (T-SOS0-1).
GROUP RELEVANT
FUNDAMENTAL
MARKETING;PUBLIC AFFAIRS
SU~JE.CTIVE.;LEVELS
SMOKING 8EHAVIOUR
July 1994
Smoking Bshaviour
Since ~he December 1993 P~ogress Report, experiments were carried ou~ with on-
I/he laser perforated gap cigars=~es. The samples (8) evaluated included 2
series of cigarettes:
r" .: Samples made Us du Maurier Ligh~ K.S. specifics=ions ~ha~ compE£se
Se ~es T
the following samples:
- a control, using porous p!ugwrap and pre-perfo~a~ed ~Ipping;
- a cellulose aci~a=e cigare=~e, laser perforated~
- a gap laser perforated cigarette where the T-section has a pressure deep
of SO ~m.:" r 7 mm length;
- a gap laser perforated cigarette where the T-se:~ion has a pressure drop
for 7 mm length.
Series ~z: s~ples made ~o Mauin6e Extra Mild K.S. specifications that
comprise ~he same fil~er consu~cuion as described.
The samples we:s selected b~ weight, pressure drop and ventilation. They were
~hen smoked unde~ s=andard smoking conditions for ~ainstream deliveries, and
a~ 35 oc and 70 co puff volumes ~o calculace elas~iolty. The effec~ of flow
rata on venti!auion was also s=udied for each cigarette sample.
The resu!=s are summarized as follows:
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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A. De~,,er~es:
Under scanda:~ smoking ¢onditi:ns, mainstream de!iveries march the control
de~iver£es.
Laser perfcra:sd cigarettes w£uh cellulose acetate or gap fi!ce=s have
higher e!asui:i:y than the cent:el, lndicaulng a relationship between
e!asulcity and on-line laser perforation.
Greater e!asulci~y was found for the laser perforated gap cigarette made
to Ma=in~e.~xu:a Mild KS specifications, perhaps due co higher venuilauion
in ~ose oigarsuues {60q as opposed to 521 foe dU M UL K~).
B. F!c'~ Rate and venci!a~io~:
Ventilation dec:eases linearly with increasing flow cane.
Ventilation dec:eases more rapidl~ with laser pe:f:raced cigarettes
wheuher the cigarettes have a ce!lulase aceca~e Or a gap filter,
Indicating char laser perforation as opposed to the use of gap filter, has
a greaser e**e.. :u on ven:ilaticn. This resul~ also suggests uha~ a~ human
smoking puff volumes (40 ~o 70 co), there should he an '. r
• nc ease in smoke
strength with :he laser perfora:sd ¢iga:e~ues due us increased dellveE!ee.
Future work will include a human smoking hehaviour s=udy concurrent with a
subjective evaluauion of ~he samples by the Expe.~-. Panel.
2. SMOK ~AG SURVEy ~
A smoking Behavlcur survey is being conducted co provide information on
¢u=Tenu smoking patterns and to evaluate the changes, if any, since the las~
survey conducted in 1981.
The s~udy includes the recording and duplication of smoking profiles f~o~
smokers recruited within ZTL. SO far, smoking profiles of 27 mmoksEs from Bhe
R & D department have been recorded and duplicaued. Analysis of the Eeeul=s
is undeF~ay.
3. ALTTRING C~G~_.~TTE C~RCUM~'~p~'~C~:
A llteracurereview is undsEway ~o investigate the effects of clrcumforence on
cigarette properules in anticipation of a study to invest!gate if and how
smokers change their hehavicur w£th dlfferenc circumference clgarst~ee.
Studies have shown a relationship between cigarette circumference and soma
factors such as par=icle character!solos in smoke, flow care BhEough the
cigarette rod, deliveries and composition of mainstream smoke and Ames
mutageniclty. FoE example, a significant effec~ of dec=easing ¢i=cumfsEenoe
is an~nc' tease in effective flow rx,o- ~hrough the rod, and as a cesul~ of
~hese changes, smokers may adjus~ c~eir smoking behaviour.
4. BI~M~.~ P~I'STOLOG~CAL RESPONSE AND M~CO~IHE U~AX~:
This is an ongoing project to invest!gate the feasibility of using h~cod
pressure measure=enOs as a means to monitor nicocine uptake. N~co~ine uptake
is affected by the availability of f~ee-base nicotine, which in turn is
affected by flow rare and smoke pH.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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BATCo document for PFSFC 1 March 1999
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RECORD TYPE:
SUB ~YPE:
SECURITY CODE:
FU~;~:~G BODY:
ORG~2~IZATION:
GROUP NUMBER:
LOCAl PROJECT. NUMBER(S):
PRO:rOT TITLE:
PERSON RESPONSIBLE:
EFFZRT:
PROJECT DESCRIPTION:
SCC~E:
DE~TH:
FDT:CT:ON:
OBJEOTIVE:
CLUSTER:
DATE REVIEW WRITTEN:
REV:EW T~TLE:
ITL CANADA
2429
Creaui=n of ~reeding Populauions from Somatic
Hybrids.
DUPIESSIS,G./~AI, D.
1994
The =~jecuive :f ~he work is Uc crea~e breeding
lines ~hrouqh :ecurrenu backcrcssing and
selecuion of numeric hybrids wiuh coE~ner=lal N__u.
cul~ivars. This work ks in
¢ollaoorauion wluh :he Delhi Research SUe:ion
of Ag=icul=ure Canada.
GROU~
FUN~;~NTAL:A~LIED/EE'/ELOPM~NT
LEAF
AGRCTECHNOLOGY
LEA~
July 1994
Creauiun of Breeding Populauions f=:m Soma:it
Hybri~s.
RE%':EW TEXT: i. Radiatioc Treanmen= of ~e debnev%
Chrz~oscme Addition 5~n~ ~n Tobacco :o Induce Trans~c=a~ion Line:
Of uhe surviving seedlings from ~he gan~na ra'$ - ~reaced addition seeds, 270
plenum -esis~anu to black roe: re: were sale=red for selflng. M2 seeds from
each were ccllecued. Ths families wiUh more resis~an: planue f:om ~he ~=ea~d
see~s ~han original addi:ion lanes a=s more likely ~= have had ~ranslocaulone
An :heir parenus. Thus, these families will ~e selecued for fu~he~ analysis.
2. C!oninu cf s~ecies sTecific DNA $;om N_..u._:a~acum~_ N__L Rustics ~d N.
d~bnevi:
m
Species specific DNA wi!~ be used in our =rojec-. for selec:ion of the
s~allesU possible alien :ransfar wiu~ ~he*gene(s) of in~erss~. Three
p ' N. ~, N. ~us~ica, and N. ~, were used in ~he initial
S SC~eSe
=~cning. One hundra~ and flfuy colonies ~iuh reccmbinan~ plasmids from
each species were is=~ausd. Screening of species smeci'ic DNA from chess
is underway using ~he Southern Blo~ ~echni~ue.
3. A Second RAPD Marker Tightly Li~ked, in Czu=linw~ ~g ~be ~u~ev 49 Gen~
f3~ Resistance ~o ~l~¢k R~O~ 8cz:
A second RAPD marker (!050 bp) was found :3 be ~i~huly linked tc the
resisuanU allele of ~he gene (in coupling:. The same RAPD maker was also
~muec~sd in N. ~ and :he ember uoba::o varieties carrying ~he gene,
b'~: no~ in ~he ~cDacc: varieties lacking :he gens. There is evidence ~ha~
:he black :oo~ ro~ resls~ancs gene in Burley 49 was from N. de~nevi.
Cleninm of ~he Butler 49 Gene f== Resis=aG7~ ~ ~ck Rogt Rot:
Use of ~he :we RAPD markers tighu!y linkel wi~h :he black =oo~ ro~
:ssisUance gene is a g:od suar~ in nhe Ic:zuion and isolauion of the
:ssis~ance genm in uz~acco gsno=es. The :=upling RAPD marksr (1050 bp)
was purified and c!one~ inuo plasmid pUCl~ in E. coll. The nex: e~su is
:C establish a uobac=c and N. da~nev~ gen=~ic llbra~/ wiuh :he resistance
-- . °
~ene
4.
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5. RAPD Fim~e.--orin~s of Canadian Tobacc~ Va~q~es:
We have ~eveloped a p=o=ocol re= exu=acUion of DNA f=cm flue-cu=e~ ==bocce
leaves. DNA from flue-cu~ed uo~acco leaves was less inu~:= khan =ha~ fEom
young f=ssh ~aba==c leaves buu did not effec= ~he resu!ua in ~he
polymerlse chain =eat=ion. Ten of Uhe 500 prime=s sc=ee~sd gene=1=~
highly repeauable polymorphism RAPD pau~erns, and finge.-~.:in=s f== each of
~he va:ie~ies have been established.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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WORK AREA 06
PROC2SS DEVELO ~."---'NT
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RECORD TYPE;
SUB TYPE:
SECUR:TY CO~E:
PUND:t:G BODY:
ORGA~I~ZAT~O~|:
GROUP NUMBER:
LOCAL PROJECT NUdgER(S,:
PROJECT TITLE:
PERSO~ RESPO~:S~BIE:
EFFORT:
P~OJECT DESCR~PT~3:~:
SCOPE:
DEPTH:
FUNCT:ON:
OB,TECTZVE:
CLUST/R:
DATE .REVIEW "'..~TTE:':
R.~'VZEW TITLE :
P--VIEW TEXT :
?
:7. CANADA
~-5352;06
GIT Process Op~imizaUion
IEVERT. G.
c~uimizauion of Green Leaf Threshlnq PlanU
p:3cesses ~hrough EeplacemanU and i~provL~en~s
=: exisuinq equipment. Phase Z; of a
=a:hinery/process upgrading prcg:~m is
==m~is~ed. Phase IIZ (e~) £s ~sLttq
i~p!emenusd. Phase IV As being planned.
13CAL
APPLIED/DEVELOPMENT
C3ST
PRODUCT. .~S/DEV
~Iy 1994
GIT Process OpULmiza~ian
~i:ious ~rocess suudles are bein~ caE~isd o~:
a) S=em dryer Cual!fi:amicn: ~=alLficau~on ~s beLnq planned wi=h a
nh~ou~hpu= of I0,C1~ ibs./h:.
C=ndi~ioni~a: mois:ure me~e= svaluaulon, op~imlza~ion of =pe=a~ion.
¢) ~eaa~a~or L=a~i~a: Opu~mizau~on.
Ba~ke~ Sizs~Sii~sr Oo~i~za:ion.
e) ~a~ V~ P~e-C~di~i3ns~ O~i~iza~ion.
f) ~als Feed $vs=~ E':aL~a~!on.
g) ~cr~qn Ma=eria! De:ec=i=n: $imco-Ramic System evaluation.
h) ~us= Co~c=~;s Sc;een~= Im=:~vemen~.
B.A.T. CONFIDENTIAL - CATEGORY h ~IINNESOTA TOBACCO LITIGATION
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BATCo document for PFSFC 1 March 1999
o.
RECOR~ TYPE : /
SUB TYPE:
SECURITY C.ODE :
FU~DI.~G HODY :
oRGANZ ZAT ~O~ :
GROUP NUMBER:
LOCAL PROJECT NUM3ER (S) :
PROJECT, TITLE :
PERS..ON RESPO.qS ZBLE:
EFFORT:
PROJECT DESC,qZPT:ON:
SCOPE:
DEPTH:
FUNCT.:ON:
OBJECTIVE:
CL~STER;
DATE REVIEW WRITTen:
REVIEW TITLE;
REVIEW TEXT:
44E
P::~ec~ Came:
H:~TLE, K.
Su~ :he in:reduction of PCL-X a~
za~ufacuurin; plan: leve~.
LC:.~I
A~I:ED/:EVEIOPMENT
P~UCT;3N
C:3T
P~::UCT .qES/:E7
P:::ec: C=me:
i) A u~:;eu packing moiscu=e of i0% was
dlfficu~ ~o mai~:ain du=ing :he summa: men:he due ~o hig~ humidi~y in ~he
produc',ion a=ea. Effa~--,s •re bei~; ma~a ~a :=naln ben=e= ~:nu==l. 2) A
~hi=d re,me= has been Insu•1!ed u: al!z~ mc:~ pEodu¢=ion ~!me re:
davelc.Dmen~a! wo=k Eelaui~g =3 a) i~c:aasin~ peE¢~n=age of ~ob•¢=o Inclusion
and b) • pr=ducu sui~ab!e for Fi~s Cuu.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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R & D SUPPORT S-'RV'rC~.S
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BATCo document for PFSFC 1 March 1999
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RECORD TYPE:
SUB TYPE:
SECURITY CODE:
FUNDING BODY:
ORGANIZATION:
GROUP NUMBER:
LOCAL PROJECT NUMBER(S]:
PROJECT TITLE:
PERSGN RESPONSIBLE:
EFFORT:
PROJECT DESCRIPTION:
SCOPE:
DEPTH:
FUNCTION:
OBJECTIVE:
CLUSTER:
DATE REVZEW WRZTTEN:
REVIEW TITLE:
REVIZWTEXT:
ITL CANADA
413
T-~01g;T-SQCO;T-~679;07
Evaluauion of ~ew P~ckage Films
DU~Z:~.R
This work is ai=ed a= pr=vidlng opnions towards
i=pr=ving the s=slf life of ~.~ pEodu~s. The
evaiuanions c=nslde= two avenues: nhose films
whi=h may have ~arrie= pEBps~u-ies equivalent to
currsnn fi!=s ~u: whose benusr sealing
characseris~ics faci!inass manufacuuring;
those films whi=h have au the onsen impr=ved
mols:urs barrier prope-~Sies. This work is
li=ksd to Dubs: acslvi~ies under uhe
"e~vi=onmenual ~-.~=s!!a".
GROUP
APPI~ED/DE'DE.LO~F~NT
July 1994
Evaiuauion QE New Package FiL~s
GLS 20 polypropy!ens film from ZCZ, prev£ousIy
reported on, was evaluated under P.D. ZI-20S6 using 120 mm O.D. cores.
Whereas ~he Uesn was generally suc:essful, scze mainly c=re-rslatsd problems
did occur. Because of a shift in emphasis to high barrier films, GLS will non
be pursued a~ nhs presens ttme. ZC: is developing a high barrier, non-
shrinking, (unlike GLS) film which shey hops t= have ready fc= trial i~ mid-
1995 wiUh a ~argsn MVTR of 0.25 g/100 sq. in./day.
The Moisnurs Retention Index (days uo lose 1% =aisture c=nten: of clgarentes
in packs an 90*F, 15% R.H.) and Array seal values have been determined for
packs wrapped in the following fi~s, all app=:xLmaue~y B0 gauge:
Reaular 8artier
Hercules (now ArT) BXT
Mobil BSRT-EXP coex
~=h Barrier
Hemcules XO-19 (now ArT UBT 368]
Mobil BSR-TX
S~p~o 5AS-C
Hoechst ZBA 20
AVE_. SEA1 VALUE
(Cent=el) 4.9
5.2
> IC0
> IC0
(Conn=ol)
7.5) +50% • IC0
v5
7.5 BXT • !CO
6.~ ÷30% • IQQ
! vs
6.5 BXT > 1C0
J
The Mobil BSR-TX film is of considerable in:erase =u: is is no: co~er:ially
available at present. The ~ecisi== to make is so is pending.
B.A.T. CONFIDENTIAL - CATEGORY I: ~IINNESOTA TOBACCO LITIGATION
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The 3SR-TX and ~he s=mowha~ lower KR~ St:pro an~ ~oechs~ films pr=duced
oxceilen: seals al:h=ugh with considerable hea~ ~is~o~:ion and s~e wr!r~ling
in the S~=pro seals. Each has been rec==~ended far Phase 2~ eva!uauion
(excsp~ Simpro which would need furuhe= ~hase Z evaluanion).
Seal Te~:inu
On su~pllsr data shss=s, seal strength values are often either no= given oE
a~e given wi~h dlffsrsnu seal ~est condi:ions or no condi:ions au all uhersby
pre¢!uding film ~o film comparison.
Our major supplier AZT use~ ~sst condi~i:ns of 2C0"F, 20 psi, 2.0 seconds for
thai: quali~y assurance testing. Dwell :ime on a high-speed packer may be
only tenths of a sac:rid therofcre seal sursnguh au shorter dwell ti~e is of
Lu~eres~.
Our initial tests a: 20 psi and 0.S seconds have produced very icw resul:s at
225"7 averaging 7 u= 60 grams per 25.4 ~ for all the regular and high ha=tier
films mentioned abcvs excep~ f3r BSRT-EXP at abou: 250. Seal strength at
250"7 and 27S°F averaged approximately 250-350 grams for all.
The $eali=g was pe~f3rm, ed on an Askco hea~ sealer which has 9 individually
conurollsd heat zones. The rssu!~s are !awer in general than those obtained
on our and AET°s Senulnel sealers. This will be lnvestigaUed.
Work will continue =: determine seal Iniuiation :emperaturss and 8ea~
strengths at various pressures and dwell times.
B.A.T. CONFIDENTIAL - CATEGORY I: ~[INNESOTA TOBACCO LITIGATION
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BATCo document for PFSFC 1 March 1999
402455275
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