A REPORT ON THE SOCIETY OF TOXICOLOGY MEETING,
DALLAS, USA, ISTH-19TH FEBRUARY 1988
By: T.G. Mitchell
4 th March 1988
Distribution:
Dr. R. Binns Copy No. 1
Mr. A.1- Heard 2
Mr. M.L. Reynolds 3,4
Dr. P.J. Dunn 5
Dr. S.R. Massey 6
Mr. R.G. Nicholls 7,8
Herm. E. Riftershaus 9
Dr. E- Kausch 10
Dr. C.J.P. de Siquaira 11
co
BATCo document for Province of BritiSh Columbia 28
October 1999


TGWJP/46D
4 th March 1988
A REPORT ON THE SOCIETY OF TOXICOLOGY MEETING,
DALLAS, USA, 15TH-19TH FEBRUARY 1988
The interest in this meeting was occasioned by the
presentation of a
series of papers by staff of R J Reynolds on their
announced development
of "the cigarette which heats, but does not burn
tobacco".
Detailed notes of the Reynolds data are provided
as Appendix A, whilst
some other reports of interest to us are reviewed
as Appendix B.
(a) Overview of the Reynolds Presentations
There was no evidence that R J Reynolds had used
this occasion for
media publicity of their development, but instead
had set out to give as
much detail of results as possible to support
independent evaluation. The
interest attracted was greater than just by
tobacco companies so it is
presumed it has succeeded in its objective of
providing a platform for
toxico;ogical peer review.
No information was provided or tendered in
response to questions
regarding samples, models or diagrams of the
device itself.
The presentations had been assembled in an orderly
fashion and could be
taken to represent part of an evaluation strategy
with similar principles to
those which we follow, e.g.:
definition of materials;
properties of materials themselves-,
properties of the total product.
(b) Nicotine
A series of four papers were given to support the
conclusion that nicotine
from the new product behaved in the same way in
the human body as
nicotine from conventional cigarettes (Paper
numbers 813, 815, 816, 817).
Monitoring of human smoking behaviour was coupled
with plasma and
urine nicotine estimations to demonstrate
comparability of uptake profile
and eiiimination, by comparison with subjects'own
brands.
(c) Glycerol
As the major additive known to be used, glycerol
has been screened by a
series of in vitro tests and 90 day inhalation
study (411, 1015). It is
questionable whether the slight reactions seen in
the inhalation study have
any great significance, but may well indicate the
need for a more
exhaustive study as 90 days is hardly a lifetime-
C:)
C)
-10
CC)
~_n
BATCo document for Province of British Columbia 28
October 1999


(d) Total Product Aerosol
Aerosol condensate has been subjected to in vitro
tests compared with
cigarette smoke (410) and 90 day whole animal
inhalation (1006-1014).
Biochemical data and aspects of aerosol chemistry
were also provided
(1063).
Presumably, to ensure data validation, contract
studies had been made of
both in vitro and in vivo work, despite the test
designs being little more
than duplication of in-house effort.
(e) Conclusions
Documented studies have been made by Reynolds
which are demonstra-
tive of their intent to prove the advances offered
by their product. These
are restricted to genotoxicity, inhalation
toxicology and nicotine response.
It is possible and even likely that other work is
in progress to extend the
studies, but at this time the biological endpoints
have been restricted to
cancer associated ones. It is known that a
volunteer study on urinary
mutagens will be described at the March EMS
meeting in Charleston,
USA, but further activities remain a speculation.
As far as could be judged, the data were well
received at the meeting, but
reflection and individual interests are likely to
combine to debate and
critique them. Potential topics could include:
(i) CO delivery - shown comparable with reference
cigarettes
in the papers;
(ii) Nicotine - if acceptable delivered in this
way, will it
offer increased possibility of misuse?
(iii) Glycerol - the dose to the consumer is much
higher than
from a conventional cigarette and it may be
questioned
whether a 90 day study is sufficient to validate
use.
There was some evidence that Reynolds may have
attempted to construct
a safety factor from testing by comparison with
cigarette smoke, as this
would be a very convenient parameter to use in
publicity.
(f) Implications
Reynolds made no product claims at this meeting
and the information on
tobacco smoke was for the greater pad confirmatory
of earlier data rather
than new. The impact on existing products is not,
therefore, particularly
great except by it being drawn together and
published by a tobacco
company.
This peer review by disinterested toxicologists at
this meeting will be of
much less significance than two other processes in
the progress of this
product - the development of the data to satisfy
regulatory review relevant
to its marketing, and the assembling and
validation of claims to satisfy lay
opinion with regard to non-injuriousness in the
light of significant CO and
wet condensate deliveries.
CD
1.0
(Do
BATCo document for Province of BritiSh Columbia 28
October 1999


A further complication of this development is the
scale of testing which it
has involved, albeit so far in limited areas. Of
itself this must escalate the
pressures to provide yet more detailed - and
independent - proof of
acceptance for future developments. The effect of
this will be to cause
increased competition for limited facilities and
place greater need for
foresight in planning their use.
CO
BATCo document for Province of BritiSh Columbia 28
October 1999


APPENDIX A:
DATA FROM REYNOLDS PRESENTATIONS
ON THE CIGARETTE THAT HEATS BUT
DOES NOT BURN TOBACCO
4 ~s-
C:)
CD
.10
00
--j
I"i
00
BATCo document for Province of British Columbia 28
October 1999


410 Doolittle, D J et al.
Genotoxicity of ci gaefte smoke and condensate
from test cigarefte
which does not burn tobacco.
Comparison of cigarettes:
Kentucky 1 R4F. 2 ultra-low tar products ex retail
market, test product
(does not burn tobacco), FTC conditions of
smoking. Pads extracted with
1 ml DMSO to 10 mg TPM. Shaken 25 min at RT,
stored at -70*C until
tested.
1. Ames mutagenesis - Revertants per plate (25 pg
upwards).
TA 98 TA 100
Activated Non-activated Activated
1 R4F 43-462 14-31 163-303
Brand 1. 4mg tar 42-421 20-39 170-333
Brand 2. 2.2mg tar 44-436 18-33 162-335
Test product 13.3mg 41-45 19-15 161-148
tar
2. SCE - Increases Over Solvent Control
Non-
activated
1 R4F 10-75;lg 33-170%.
Brand 1 15-75 Vg 12-87%
Brand 2 15-75 pg 25,4,64,113
Test 25-100 pg 0,0,0,0
100 pg toxic. Dose levels v'.
Activated
150-399 lig 35,51,92,70
32,45,29,48
21,32,45.44
0,2,0,0
CD
1.0
00
BATCo document for Province of British Columbia 28
October 1999


3. Chromosomes (% with alterations)
Non-activated Activated
1 R4F 125-200gg 17.5,33,32,62 150-300gg
2.5,3,8,23.5
Brand 1 9,45.58,58 1.5,1.0,4.5,
10.5
Brand 2 27.5,60.5,65, 2,9.5,23,29
48.5
Test 1.5,1.5,3.0, 1,0.5,0,0
0.5
4. UDS - Increase in Net Nuclear Grain
I R4F 25,50,75,125,150 0,8.2,8.3,13. 1,Toxic
49
Brand 1 25,50,75,100,125 7.3,4.2,11.5,13.5,Toxic
99
Brand 2 25,50,75,100 119 5.5,10.9,18.5,Toxic
Test 25,50,75,100,125 0,0.5,0,0,0.1,Toxic
119
5. Mammalian Mutagenesis - Mutant Frequency / to,
cells
(negative in all cases)
Non-activated Activated
100-300 pg 38,54,43,63,69 44,39,53,51,50
4 ~1j.
CZ)
CD
110
c0
L-4
CD
BATCo document for Province of British Columbia 28
October 1999


411 Lee, C K et al
Genotoxic testing of glycerol in vitro.
Ln 3~U Tests of Givcerol
1. Ames test according to Maron & Ames, 1983. S9
~) 5%.
2. SCE - Method of Perry & Wolff, 1974. 2h
activation, 25.5h non-activa-
tion.
3. Chromosome aberrations - Method of Galloway et
aL, 1985.
CHO cells, 10 & 14h exposure with no recovery time
for
non-activated system;
2 h + 10 & 1 4h recovery in activated system.
4. HGPRT Gene Mutation Assay - Method of O'Neill
et aL, 1977, using
CHO KI BH4 cells.
5. UDS - Method of Mitchell & Mirsalis. CID male
rat, 18h.
Results for SCE
Non-activated Activated
Negative Control 6.82 8.14
Positive Control 22.64 23.04
Glycerol 200-1000 gg Iml 6.88-7.44 8.4-9.6
Results for HGPRT
Non-activated Cloning Mutants Mutants per ial
cells
efficiency
Negative Control 86 2 2
Solvent Control 76 6 7
Positive Control 77 178 200
Glycerol 100-1000 gg 65-80 6,0,2,3,7,12
7,0,3,4,9,15
Activated
Negative Control 4
1 5
Solvent Control 75-84 2 2
Positive Control f 122 153
Glycerol 100-1000 pLg 79-87 0,1,1,4,20,5
0,11,11.5,24,6
CD
CD
1.0
co
-14
LJQ
BATCo document for Province of British Columbia 28
October 1999


8
813 Bates, L E et al
Monitoring nicotine in pharmacokinetic studies
Two methods of measuring nicotine in vivo were
compared in rats and,
overall, found to give comparable results. Data
points for individual
animals did show apparent substantial variation
between methods, but
these were believed to represent sample to sample
variation.
Summarised results were provided for plasma
haff-life of nicotine in rats
following a single Vv dose of 2.2 pmkg-,
1/2 life (min) n mole
Animal
A B A B
1 62.3 78.3 163 139
2 78.9 100.5 255 272
3 66.6 63.8 229 193
4 109.7 95.3 220 239
5 168.5 167 295 295
Mean 97.2 101 232 228
Method A: Ramona LS-4 radiometric HPLG
Method 8: HPLC fraction collector
3
Nicotine
n moles
(mean)
INI
50 350
Time
C:)
%~O
CC)
rQ
BATCo document for Province of BritiSh Columbia 28
October 1999


815 Griffiths, D W eta[
Human smoking behaviour and cigarette yields
Used 10 port machine, driven simultaneously.
(a) 5 subjects normally smoking full flavour low
tar brand:
VVTPM Nicotine mg Tar mg
mg
Same brand 11.62 0.826 8.45
(mean)
Range 5.9-17.6 4.54-11.3
FTC method 0.62 8.5
(b) Puffing behaviour, 19 subjects, using regular
brands:
Mean s.d. Range
Number puffs 12.5 4.3 8-23
Volume 44.1 12.1 12.5-63.2
Max. draft pres- 372 100 193-516
sure (nww,o)
Puff duration 1.84 0.49 0.9-2.6
Frequency (secs) 33.2 13.4 14.1-61.4
CD
CD
co
BATCo document for Province of BritiSh Columbia 28
October 1999


10
(c) Individual nicotine yields, regular brands and
'TEST' cigarette
Subject FI-Cnicotine Nicotine(mg) `TEST'nic-
(mg) regular regular brand otine (mg)
brand
1 0.62 0.67 0.47
2 0.62 1.07 0.91
3 - 1.26 0.76
4 0.62 1.15 0.75
5 0.65 0.88 0.88
6 - 0.86 0.80
7 1.2 1.31 1.00
8 1.14 2.49 1.06
9 0.62 0.79 0.89
10 0.70 1.49 0.88
11 1.14 1.44 0.68
12 0.92 1.98 0.95
Conclusion:
Individuals smoking a test cigarette which heats
but does not burn tobacco
exhibited different puffing behaviours from those
whilst smoking regular
brands. Differences in nicotine yields were also
observed which could not
be determined from measurements of the puffing
patterns without ma-
chine replication of the smoking profiles.
BATCo document for Province of BritiSh Columbia 28
October 1999


816 Robinson, J H et al
Nicotine yields during human smoking
Procedure:
11 pm previous night - no more smoking.
8.30 am next day - start test.
Cannulate vain.
Smoke 1 cigarette every 30 min, up to 7
cigarettes.
Monitor puffing, breathing.
Baseline samples: -2 and 0 mins.
Cigarefte-related samples 1-6:5.5, 7.5,10,15 & 30
mins.
Cigarette-related sample 7: 5.5, 7.5, 10, 15, 30,
45, 60,
90,120 & 180 mins.
Subiects:
4 males, average age 28, height 176cm, weight
76kg.
Cigarettes per day: 30
Regular brand IFTC figures: Tar 14.2. nicotine
0.96, CO 13.1, CO, 43.3
Mg.
Average puffing parameters
Number puffs: 11.6
Puff volume: 53.9 ml
Puff frequency: 28 secs
Puff duration: 2.35 secs
Total puff volume: 615 ml
Time alight: 299
Average max. flow, .1 41.7
Average max. draft, nintH.0
460.6
Fyoerimental clQarette: deliveries
Nicotine mg
Go mg
CO, mg
IFTC HMSM
0.71 1.7
12.5 24.6
36.5 58.3
Average breathing patterns
Number of breaths: 11.5
Inspiratory volume: 713 ml
Inspiratory time: 1.7 secs
% Inspiratory time: 36.2
Expiratory volume: 778 ml
Expiratory time: 2.79 secs
CD
(Z)
%D
cc
BATCo document for Province of BritiSh Columbia 28
October 1999


12
Plasma nicotine over 7 cigarettes ( ng
Subject: 1 2 3 4
Cigarette
1 355 638 403 246
2 394 561 513 346
3 350 640 610 688
4 339 722 636 593
5 427 813 757 642
6 424 765 737 753
7 437 792 941 731
Average 389.6 704 657 571.3
C)
1%0
co
BATCo document for Province of British Columbia 28
October 1999


13
817 de Bethizy, J D el al
Nicotine availability from cigarette which does
not burn tobacco
51 y* design:
Controlled diet 6 days
Tobacco withheld days 3-6
Nicotine bitartrate infusion day 6: 1.91,,/
/kg/min, 10 min
Blood samples from cannulated veins:
-2, 0. 2, 5, 7.5, 10-90 mins
2, 4, 5, 6, 8 hours
Urine samples: 0, 1.5, 3, 4.5, 6, 8 hours
Smokers:
Average age 35.2 years, helight 178.3 cm, weight
85.9 kg.
Average cigarette consumption 26.6/day, years
smoked 15.9,
Brand FTC tar 13.1 ;L 3.8 mg, nicotine 0.81 mg.
Smokina Study:
Tobacco withheld days 3-5
Blood samples taken via cannula
1 cigarette smoked per 30 minutes, up to 7
Products ComDared:
Reference cigarette: Tar 8.8 mg (83.3%). Nicotine
0.66 mg (6.3%),
Water 1.1 mg.
'TEST' cigarette: Water 6.8 mg (55.21/6), glycerol
4 mg (32.4%).
Propylene glycol 0.3 mg (2.4%), Nicotine 0.34 mg
(2.8%),
Other 0.9 mg (7.3%).
Results of i/v nicotine infusion (12 subjects) -
mean response.
"A\
too
CD
C:)
c0
--J
BATCo document for Province of British Columbia 28
October 1999


14
Smokino -Studies: 10 inhalers - mean plasma
nicotine
Curves were same shape and general response for
reference and 7EST',
with latter giving rather lower response in first
run, but a second one being
closer to the reference.
In two non-inhalers, responses were flat for both
reference and 7EST
products, staying at around 2 ng/ml throughout the
test period.
Urinary nicotine values also showed an increased
response in the second
run with the 7EST'product:
'%edt.' Or..A. Tone
^80'0t'~-e r<
L
.2 -S'
F t-i,
3 : 2. C4 39 d~7
0
CD
CD
.10
CO
L-4
00
BATCo document for Province of BritiSh Columbia 28
October 1999


15
Nicotine absorbed from 7th cigarette was estimated
to be:
Reference: 1024 gg ( 315)
TEST : initial - 579 pg ( 198) (56% of reference)
: after 39 days use - 705 pg ( 211) (69% of
reference)
Absorbed vs. yielded nicotine (7th cigarette)
Referenc Test
e
Initial Post 39
days
*Yielded ( pg 1659 754 780
Absorbed (pg 1024 579 705
% Absorbed 63 80 87
'By human mimic smoking machine
Conclusion:
New cigarette produces plasma profile very similar
to reference.
Nicotine yield from both was proportional to the
amount of nicotine yielded
by cigarettes when subjects smoked each cigarette
for the first time.
There were no differences in elimination of urine
between reference and
TEST.
C)
~.O
CO
-_J
BATCo document for Province of British Columbia 28
October 1999


16
1006 Hayes, A W et al
90 day study. experimental design
Grows:
30 male, 30 female Sprague Dawley rats in each
exposed nose-only 1 h
per day, 5d per week, 13 weeks. Smoke exposure
Low, Medium, High,
doses based on nicotine.
Cioarettes:
Reference (RJR) matched TEST (cigarette which does
not burn tobacco)
on WTPM, CO, nicotine; therefore equivalent
nicotine and also WTPM,
CO. Use of nicotine as matching parameter based on
plasma nicotine
availability.
Reversibilitv:
High and sham groups, 10 animals of each sex, with
7 week period after
conclusion of smoke exposure.
Desionations:
MC - Machine control
RC - Room control
TLD - TEST low dose - 5 ggli nicotine - Reference
low dose RMD
TMD - TEST medium dose - 15 pgll nicotine -
Reference medium dose
RMID
THD - TEST high dose - 30 ggli nicotine -
Reference high dose RHD
Cigarette data (FrQ - both liftere
Puffs WTPM mg CO mg Nicotine ~jg
TEST 8 10.1 11 465
Reference 7.1 9.7 12.8 480
(made by RJR)
IR4F 9.2 10.8 11.6 800
(not used in this
study)
Smoke machine allowed full operation of filters.
Inhalation device:
Nose only, age of smoke at nose 1-2 secs, no
rebreathing. Continuous
flow of aerosol at each port at rates well in
excess of ventilatory C)
requirement to minimise breath holding. CD
%0
co
_~j
41.
C:)
BATCo document for Province of BritiSh Columbia 28
October 1999


17
Bloloaical endooints:
Blood CO +55", weeks 1. 4. 8.12, as check that
potentially fatal level not
reached.
Nicotine +55", weeks 2, 5, 9, 13.
Minute ventilation (also 10* pre-exposure) weeks
1. 5, 9, 13, throughout
exposure.
Target dosimetry achieved, particle sizes
identical. Aerosols were inhaled
differently because reference caused decrease in
minute ventilation.
TEST inhaled in much larger quantities with
resultant higher plasma
nicotine. A warming burner was required for use
with the TEST cigarette.
CD
CD
I'D
CO
BATCo document for Province of BritiSh Columbia 28
October 1999


18
1007,lames, R A et al
90 day study: smoke chemistry
'C"O w
lei
Ow rfte.
M 0"1 Tdf VA
co,4 -rg*L
4,
ea
e
Chamber nicotine - TEST and reference essentially
superimposed.
&.0 L4
Weeks.
0-2-
"0 -
C:)
C:)
%~O
CO
BATCo document for Province of BritiSh Columbia 28
October 1999


19
1008Coggins, C RE et a/
90 day study. Blood composition.
Blood Collection: retroorbital sinus under 701/6
CO in air anaesthesia.
CO: No difference between reference and TEST weeks
1,4,8,12, or
between sexes, n = 37-40.
COHb % Low Medium High
TEST 12.2 25.2 40.6
Reference 9.46 22.5 39.4
Nicotine (ng/ml), n = 60-70
COHb % Low Medium High
TEST 42.4 126 238
Reference 14.0 29.3 55.5
Cotinine (ng/ml)
COHb % Low Medium High
TEST 44.9 103 158
Reference 12.1 20.2 28.4
Cotinine: nicotin e ratios
COHIJ % Low Medium High
TEST 121 87.9 75.3
Reference 97.8 81.4 56.9
Nicotine inhaled and retained higher in test.
Plasma nicotine in part due to
reference depression of minute volume. These
calculations reduced
factor for test over reference from 4.53 to 1.96.
Gotinine values and ratios showsmetabolism simile
for TEST and refer-
ence. Plasma nicotine and cotinine at necropsy
16-24h after last
exposure essentially same (claimed but no figures
provided) with no
evidence of accumulation.
High blood COHb relevant to increased heart
weights. Clinical pathology
endpoints - no changes, no value in
discriminationi
C)
C)
%4
CO
BATCo document for Province of BritiSh Columbia 28
October 1999


20
BLOOD CAn130XYHEM0GLO81M CONCENTRATICtrS (SEXES
COMBINE01 IN SA PLEr TAKEN AFTER 55 MINUTES OF
EVIOSUPE TO
S14OKE FROM TEST AND REFERENCE CIGARETTES.
A5
30
Z
TLO T.0 THD PLO RMO
PLASMA Nrcorpe CONCE-RATIONS JsF)cES
COMBI NED) IN SAMPLES TAKEN AFT R 55 MINUTES OF
ExrOSURE 79
SMOKE FROM Te ST Atio REFFR9MCE CIGARETTES.
300.
200
100
0
TLD TMO 7.0 PLO AMD A.0
P ATIOISNIP ET.EE. ICOTT "E PPE-Sr.1E. A
C,
.~.ULTING PLASMA NICOTIME CON .,RATION. FOR BEST
NO
AEFEREtACE CIGAPETTE~.
250
200
150- TEST
100.
c0
BATCo document for Province of British Columbia 28
October 1999
REFERENCE
so 20 3.
I.d


21
1009 Mosberg, A T et al
90 day study: Minute ventilation
Whole body plethysmography. Flow signal analysis -
Buxco irritation
monitor. Four subjects simultaneously.
WTpM NICOTINE co
MEAN SID N MEAN SID MEAN SID
TLD 115 13 68 5.23 0.75 144 27
TMID 325 24 68 15.2 1.8 394 75
THD 604 34 68 30.6 3.2 698 133
RLD 138 9 68 5-18 0.7 160 14
RMID 411 20 68 15.2 1.3 459 35
RHD 795 25 68 29.8 2.1 864 60
Calculated Total Inhaled Mass:
WTPM NICOTINE CO
TLD 1021 46.4 1406
TMD 2944 137.7 3926
THD 6306 319.5 8016
RLD 861 32.3 1098
RMD 1701 62.9 2098
RHD 3100 116.2 3706
C)
C)
1.10
00
BATCo document for Province of British Columbia 28
October 1999


22
101OAyres, PH et al
90 day study: Body and organ weights
Reference and test had same effect on body weight.
Restraint causes stress - depression in sham
smoked.
Even low dose aEproached operational definition of
maximum tolerated
dose as defined y body weight 90% or less of
control.
Increase in heart we' hts reflected concentration
of CO - similar effects
r
with CO alone in 14 Tay study - 527 ppm. CO caused
significant increase
in heart to brain weight ratio.
All observed body and organ weight changes in
smoke exposed groups
were same as sham exposed groups at end of
recovery period, indicating
that these changes regress and are not
progressive.
C)
co
--4
X:..
BATCo document for Province of British Columbia 28
October 1999


23
1011 Burger, G T et al
90 day study: histopathology
Scoring, etc.
Nasal sections - levels 1, 11, 111 and IV of Young
used.
Lesion scoring - 1 minimal change, 2 mild change,
3 moderate, 4 marked,
5 severe. TL total examined.
General:
Most changes reversible within 7 week recovery
period.
Nasal:
Reference - Chronic active inflammation,
epithelial hyperplasia, squamous
metaplasia and goblet cell hypertrophy 1.
11 - Atrophy of olfactory epithelium in high dose
reference. Changes in
reference high dose only partially reversible.
Larynx:
Reference - Squamous metaplasia ventral epithelium
all reference,
throughout organ. Normal "pseudostrafified"
cuboidal to low columnar
epithelium transformed to stratified squamous
epithelium with typical
progression of mat of basal cells to flattened
layers of keratinized desqua-
mating cell remnants.
Test - Minimal squamous cell metaplasia only in
high dose in a precise
area at base of epiglottis.
Conductina airways:
Reference - Increase in Alcian Blue/PAS +ve goblet
cells. No difference
after reversability period.
CD
C)
.10
00
BATCo document for Province of BritiSh Columbia 28
October 1999


24
MALES FEMALES
NASALI
- 1 2 3 TL - 1 2 3 TL
MC 18 0 2 0 20 20 0 0 0 20
THD 20 0 0 0 20 19 0 0 0 19
RHD 4 2 14 0 20 2 7 9 1 19
After reversibility period
- 1 2 3 TL - 1 2 3 TL
MC 10 0 0 0 10 10 0 0 0 10
THD 10 0 0 0 10 10 0 0 0 10
RHD 8 1 1 0 10 8 1 1 0 10
NASALII
- 1 2 3 TL - 1 2 3 TL
MG 20 0 0 0 20 20 0 0 0 20
THD 20 0 0 0 20 19 0 0 0 19
RHD 14 3 3 0 20 13 1 5 0 19
After reversibility period
- 1 2 3 TL - 1 2 3 TL
MG 10 0 0 0 10 10 0 0 0 10
THD 10 0 0 0 10 10 0 0 0 10
RHD 10 0 0 0 10 8 0 0 0 8
C=)
C)
1.0
CO
CO
BATCo document for Province of BritiSh Columbia 28
October 1999


25
1012 Wehner, A P et al
90 day Battelle study in rats.
Groups:
34 Sprague-Dawley rats of each sex per group.
Cigarettes:
1 R4F and TEST cigarette which does not burn
tobacco at doses of 0,
0.12,0.35 and 0.64 mg/l TPM, 1 hour/day, 5
days/week, for 13 weeks.
WTPM Nicotine CO MMAD
mg 99 mg IL
I R41F 0.122 0.013 6.4 0.9 0.146 35 0.55
0.347 0.025 19.9 1.8 0.413 96 0.61
0.632 0.035 37.7 3.4 0.733 102 0.60
TEST 0.119 0.015 2.5 0.5 0.187 43 0.68
0.349 0.03 8.3 1.2 0.505 98 0.66
0.62 0.056 16.4 2 0.870 117 0.63
Chemical:
No effects by TEST or reference.
Bodvweiaht:
Lower than treated controls.
Lower than sham in male medium and high reference
and high test and in
female high test.
Haemoglobin elevated, also VPRC in female high
test group.
ReSDiratorv freauencv depressed in mid and high
reference groups.
CO
BATCo document for Province of BritiSh Columbia 28
October 1999


26
1013 Henne, R A of at
90 day hamster study.
Cioarettes:
1 R4F and TEST cigarette which does not burn
tobacco, target exposures
0.12,0.35 and 0.64 mgA WTPM.
Smoke dgl%
WTPM Nicotine Glycerol CO MMAD
mg\l vgll gg/I 11
1 R4F 0.123 0.013 7.2 0.5 11.1 141 141 0.68
0.323 0.04 17.9 2.2 28 5 432 0.65
0.665 0.007 41.2 0.7 61 851 0.56
TEST 0.124 0.001 3.4 0.4 89 4 125 0.75
0.358 8.6 0.8 251 3 394 0.74
0.657 0.011 17.4 0.5 443 2 772 0.75
Histooatholoqv:
REFERENCE TEST
Squarnous metaplasia, acanthosis, No effect on
larynx.
hyperkeratosis of epithelium at base
of epiglottis, suppurative laryngitis.
Aggregates of macrophages in pul- Aggregates of
macrophages in
monary alveoli largely regressed + 6 pulmonary
alveoli, + 6 weeks re-
weeks from end of exposure. Nu- gressed. Pigment
rarely observed
merous macrophages containing pig- at highest
concentration.
ment at highest concentration.
Hyperplasia and squamous metapla- No effect on
nose.
sia of nasal respiratory epithelium.
Degeneration of olfactory epithelium.
All lesions regressed either partly or All lesions
completely regressed 6
completely 6 weeks after finish of weeks after
exposure.
exposure.
-Ch.
C)
C=)
%.0
CO
Ln
CD
BATCo document for Province of British Columbia 28
October 1999


27
INCIDENCE OF LESIONS
+ 6 weeks
TEST Reference
Un- Un-
treatec TEST Reference
treatec,,
Sham .12.35.64.12.35.64 Sham .12.35.64.12.35.65
30 29 28 30 29 30 30 30 20 19 20 20 20 19 20 20
LARYNX
Squamous 0 0 0 0 0 29 29 30 0 0 0 0 0 0 1 2
metaplasia
Acanthosis 0 0 0 0 0 29 29 30 0 0 0 0 0 0 1 4
Hyperkera-
tosis 0 0 0 0 0 14 12 19 0 0 0 0 0 0 0 1
Inflarnma- 0 0 0 0 0 26 26 20 0 1 0 0 1 1 4 8
tion
_UNG
PAM aggre- 4 8 13 11 18 15 20 24 2 2 4 0 0 5 2 6
gates
NOSE
Respiratory
epithelium
iyperplasia 1 0 1 0 0 2 30 30 0 0 0 0 0 0 0 0
Squamous 0 0 0 0 0 0 2 14 0 1 0 0 0 0 0 0
metaplasia
Olfactory
epithelium
Degenera- 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2
tion
C:)
CD
1.0
co
BATCo document for Province of British Columbia 28
October 1999


28
Larynaeal histoDatholoov - Reference only -
Incidence (%)
13 weeks 13 + 6 weeks recovery
Concentration 0.12 0.35 0.64 (0.12) (0-35) (0.64)
WTPM
Number 30 30 30 19 20 20
Squamous meta-
plasia
Minimal 20 10 7 0 5 5
Mild 63 70 23 0 0 5
Moderate 13 17 43 0 0 0
Marked 0 0 27 0 0 0
Acanthosis
Minimal 20 10 7 0 5 15
Mild 63 70 23 0 0 5
Moderate 13 17 43 0 0 0
Marked 0 0 27 0 0 0
Hyperkeratosis
Minimal 43 40 33 0 0 5
Mild 3 0 27 0 0 0
Moderate 0 0 3 0 0 0
Marked 0 0 0 0 0 0
Suppurative In-
flammation
Minimal 37 33 3 5 10 15
Mild 50 53 43 0 10 20
Moderate 0 0 20 0 0 5
Marked 0 0 0 0 0 0
co
_j
Lri
(Ij
BATCo document for Province of British Columbia 28
October 1999


29
1014 Gerald, L e1 al
Extended inhalation exposures
An attempt was made to develop a procedure to
increase smoke exposure
in cigarette studies by extending the riod per day
from 1 to 6 hours. At
a level of 0.3 mg TPM11itre from 1 R4rigarettes
for 6 hours, GOHb
concentrations in rats were similar to those
obtained after 0.6 mg TPM/litre
for I hour. However, marked depression of body
weight in the extended
exposure group indicated poor tolerance to this
regime after 10 days and,
therefore, its inapplicability to a sub-chronic
study.
CD
110
CO
U'l
BATCO document for Province of BritiSh Columbia 28
October 1999


30
1015 Greenspan, B J et a/
Humectant aerosol study
2 week study Glycerol
2 week study Glycerol
mixture
13 week study Glycerol
Mean body weights: no effect
Laryngeal histopathology:
2 week study
Sham 4 mg
Number 10 14
examined
Squamous
metaplasia
Minimal 1
Mild 0
Target conc. Mean conc. MMAD ja
mg/l
1 1.01 1.46
2 2.01 1.45
4 4.05 1.36
1 0.99 1.43
2 1.99 1.42
4 3.87 1.67
0.033 0.033 1.09
0.165 0.166 1.49
0.660 0.662 1.61
13 week study
Sham 0.033 0.165 0.66
25 19 20 21
6 2 1 4 10
7 0 0 0 1
0.033 result similar to sham. Clara cells from
high and sham did not show
any proliferation of smooth endoplasmic reticulurn
as found in mice given
glycerol orally-
.P.
C)
C)
1 ~0
00
Cn
BATCo document for Province of BritiSh Columbia 28
October 1999


31
1063 Chang, M K et al
Cytochrome P-450
Delivedes Reference TEST % Reduc-
(per cigarette) tion
in TEST
Benzo(a)pyrene ng 9.2 0.1 98.9
Benm(a)anthracene ng 10.5 0.12 98.9
Nitrosamines
1
'INN ng
101
6.6
93.5
14 NK ng
84 3.8
95.5
MATB ng 114 11.4 90.0
Pherol ;,Lg 15 0.5 96.6
Cate-.hol lig 79 2-6 96.7
Aldenydes
.Acetaldehyde l.Lg 627 34 94.6
Acrolein gg 73 10 86.3
Formaldehyde Rg 17 13 23.5
Inhaled doses - rats, 60 minutes.
Total Inhaled Dose Plasma Nic- COHb
otine
Nicotine WTPM ng/ml %
99 mg
TES-L 207.03 4.98 269.3 67.9 52
Reference 130.71 1.92 112.3 25.4 28.2
Effect on P450 Protein Cytochrome Cytochrome
mg/g lung P450 C reductase
nmole/m g nmole/mg
protein protein/min
Sha-n 3.42 0.1 26.14
Reference 4h 3.23 0.08 31.88
ReWence 24h 3.01 0.13 34.21
TES'T Or 3.33 0.08 24.92
TEST 24hr 3.1 0.13 23.82
Room control 3.35 0.13 26.80
Phenobarbitone 2.92 0.13 29.67
3-rrethylcholanthrene 3.19 0.11 35.7 Xb.
resut: no significant effects. C:1
CD
00
Cri
~_n
BATCO document for Province of BritiSh Columbia 28
October 1999


32
Effect on pulmonary P450 monooxygenase:
BaP hydroxylase Ethoxyresorufin
pmole/mg/min 0-deethylase
pmole/mg/hr
Sham 56.58 32.77
Reference 4h 149 207.03
Reference 24h 107 104.4
TEST4h 64.65 59
TEST 24h 60.8 28.8
Room control 76.8 35.7
Phenobarbitone 58.3 33.5
3-methylcholanthrene 497.2 527
Conclusion: reference caused significant induction
of enzyme activity
whilst TEST similar to controls.
C:)
CD
%0
C7%
BATCo document for Province of BritiSh Columbia 28
October 1999


33
APPENDIX B
OTHER NOTES ON PAPERS PRESENTED AT DALLAS MEETING
Jz~.
C:)
C:)
.10
c0
-~j
Ln
-Ii
BATCo document for Province of British Columbia 28
October 1999


34
(a) Cigarette smoke
Three reports examined aspects of cigarette smoke:
412 - Ames muta-
enicity;
96 - depression of immune response; 637 -
haemoglobin adducts in
9
smokers.
(b) Nicotine
A paper on dermal nicotine absorbtion (489)
nominally related to pesticide
uptake. The half lives determined by this mute may
be of interest for
comparison.
(c) Nitrosamines
A study purported to relate toxic effects of
nitrosamines to their carcino-
genic action (573).
(d) Genetic endpoints in respiratory tissue
Two presentations (6, 678) indicated that some
progress is being made on
short term responses in respiratory tissue, and
can be used to assess
interactions between materials.
(e) Symposium session
Combined Exposures in Inhalation Toxicology.
Essentially a position
review. The accompanying notes were to record
points of possible
relevance to cigarette smoke.
(f) Agricultural chemicals
Note was made of 5 (571, 702, 699, 705, 405) of
some interest to
ourselves.
(g) Vinyl acetate (646, 658, 969)
Shown to be carcinogenic at high doses in animals
by inhalation but not
by mouth.
(h) Cournarin (135)
A biochemical study from BIBRA identifying the
mechanism for differential
toxicity of coumarin and dihydrocournarin in rat
liver.
C:)
C:)
%0
co
Cn
Co
BATCo document for Province of BritiSh Columbia 28
October 1999


35
412 Chortyk, 0 T et a/
Mutagenicity of low tar cigarette smoke
Ames activity of a range of US cigarettes was
compared with that of a
non-filter high tar King Size product (23 mg).
Methodology used was to collect smoke using a
modified Borgwaldt
machine, using 2 acetone-chloroform traps in
series for mainstream and 3
traps for sidestream.
VACWV
Tea,jo v
AO.P.
Solvent was removed under vacuum and the weighed
condensate
dissolved in DIVISO and tested against 2 strains -
TA 1538, TA 100 - at
concentrations from 50-200 gg ml
Test system:
0.5 ml S-9, 0. 1 ml organism suspension, 0. 1 ml
test solution, mixed, held
37* 20 min, mixed with 2 ml top agar - plated to
minimal medium,
incubated 37' 48h.
Results obtained varied from 60 revertants up to
270 for 200 ~tg dose for
mainstream smoke, compared with 200-300 revertants
at 200 Ag for
sidestream.
Results given were:
C)
C)
%D
CO
Ln
BATCo document for Province of BritiSh Columbia 28
October 1999


36
Relative Mutagenicity
Tip perf- TA1538 TA100
Cigarette FTC oration
size (Mg) (rows) IVIS SS IVIS SS
King 1 2 64 137 31 108
100 1 2 98 122 103 103
100 2 1 67 151 54 136
100 3 1 42 115 34 120
King 3 6 81 140 74 134
King 3 - 29 161 54 110
100 4 6 73 116 80 92
100 5 - 75 132 92 153
100 5 2 113 108
100 6 - 80 105 89 107
100 6 1 48 124 64 138
100 7 1 59 146 65 143
King 8 - 84 120 72 100
King 8 - 58 'I'll 73 102
King 9 1 72 108 60 86
100 10 - 83 100 52 106
CD
c0
_,j
C:)
BATCO document for Province of BritiSh Columbia 28
October 1999


37
596 Chilukuri, R et al
Effect of cigarette smoke on immune response
The effect of cigarette smoke on immunogenic
response was made using
Kentucky reference cigarettes as smoke source with
a dose calculated
equivalent to I pack per day for man (? 6mg kg-,
day.,). Details of smoke
exposure were limited and monitoring of this was
by COHb and chamber
TPM only.
Studies were made with anti-sheep RBC as the
immune response
measured, with effect expressed as PFC 10, (plaque
forming counts?).
Antigen Smoke Tissue
Challenge Exposure
(weeks)
Intratracheal 16 LAL
+ footpad injec- PLN
tion
PFC.10-- % Inhibition
by smoke
1
Sham Smoked
772 506 35
1182 1250 0
35 LAL 1718 744 57
PLN 1274 672 48
Footpad only 18 Spleen 686 640 6
39 Spleen 600 358 41
LAL - Lung Associated Lymph node; PLN - Peripheral
Lymph Node
Subsequent experiments showed no difference in T
and B cell distribution
from smoke exposure, no change in T cell subsets,
nor on macrophage
function. There was no evidence of altered
response to T cell mitogens
(PHA, Con A).
A differential response to Brucella abortus
antigen was also shown. It was
concluded that effect of smoke was due to
preferential effect on B cells.
C)
110
CO
BATCo document for Province of BritiSh Columbia 28
October 1999


38
637 Latriano, L et al
Ethylene oxide and 4-aminobiphenyl haemoglobin
adducts in
smokers
A study of ethylene oxide and 4-aminobiphenyl
haemoglobin adducts has
been made in smokers and non-smokers. The data
summary is as
follows:
Adduct
Mean value
Ethylene oxide Range
Number
Mean value
4 Aminobiphenyl Range
Number
Smokers Non-smokers
596 264 46 27
188-982 26-101
13 7
142 38 28 13
75-178 11-44
13 7
Whilst the data showed an apparent clear
difference between smokers
and non-smokers, there was no obvious dose
response with nominal
variations in cigarette consumption.
CD
-10
CO
BATCO document for Province of BritiSh Columbia 28
October 1999


39
489 Hall, L L et al
Percutaneous penetration of nicotine
This EPA study was made using labelled nicotine as
a model to compare
the effect of age of animal on rate of uptake. A
related paper examined
methylarsenatesl
100 or 200 gl of an acetone solution of nicotine
was applied to clipped
back areas of 33- and 82-day old rats, to provide
a nicotine dose of 285
nmol cm, over 2.3% of body area.
At 6h, penetration of activity was 46% in the
young rats, compared with
26% for the adults. Half-lives for recovered
activity were also significantly
extended in adults:
Young: 75% of recovered activity, half life 1.1 h
24% of recovered activity, half life 4.7h
Adults: 72% of recovered activity, half life 2.6h
28% of recovered activity, half life 9.3h
Residual body burden at 48h was 7% in adults
compared with 3. 1 % in
young.
C)
C)
I'D
CO
ON
BATCo document for Province of BritiSh Columbia 28
October 1999


40
678 Cosma, G N et al
CNA protein crosslinks following formaldehyde and
benzo(a)pyrene
exposure
A model system using rat tracheal implants has
been employed to
examne the interactive effects of formaldehyde and
BaP. The end point
is DN'k-protein crosslinking. which is measured
against a control response
in whdh the DNA is treated with 300 rads to give a
standard elution
profike. The degree to which this profile is
altered is a measure of
DNA-Protein cross-linking (DIRC).
Formaldehyde gives a concentration dependent
response in this system
with detectable results at 0.005% and maximum at
0.2%. This is
independent of using multiple or single exposures.
At the highest dose,
remcial of the DPC requires at least 3 days
recovery.
If the-9 is pre-exposure to BaP, the effect of
formaldehyde is diminished,
but is eventually lost with increasing
concentrations of formaldehyde.
An irEeresting pathological response to use of the
2 agents was seen, with
an in7.eased epitheliall thickness compared to
either alone. This was
acco-npanied by focal areas of cell atypia with
bizarre nuclei and abnormal
mitoti; figures.
C)
CD
I'D
00
BATCo document for Province of BritiSh Columbia 28
October 1999


41
6 Bond, J A at at
DNA adducts and carcinoma following diesel soot
exposure
30 month exposures of rats to diesel soot
concentrations up to 7mg m, for
7 h.dFirl led to adenocarcinomas in the lung
parenchyma at 24 months
The a-stribution of DNA adducts in the respiratory
tract was examined
after 12 weeks exposure to diesel soot at 10 mg
m-3, 7h day', 5 days
week-,- In broad terms there was a relationship
with ultimate carcinoma
distributon. Small numbers of adducts were found
in nasal tissue,
negliable numbers (1 per 109 bases) in mainstern
bronchi whilst about 200
per 1 CF1 bases were recovered in lung parenchyma.
Two major adducts
were :;resent in lung parenchyma, one of which is
provisionally identified
as 130 diol epoxide adduct. Pulmonary macrophages
also contained
signify-ant numbers of adducts compared with
control non-exposed, but
their pattern differed from that of the lung
parenchyma samples.
CD
C)
%D
c0
__4
Un
BATCo document for Province of British Columbia 28
October 1999


42
Symposium Session
Combined exposures in inhalation toxicology (no
abstracts provided)
This session was rather disappointing in being
little more than a review of
current knowledge. Indeed, its most invigorating
moments came with an
attack from the floor of a presentation on
strategies. essentially
condemning current procedures and the people
involved for rigidity in
outlook and an obsession with use of the rat.
Notes on individual
presentations were as follows:
Kennedy,A
Radiation and chemicals. This was mainly a review
of tobacco
smoke/smoking interactions.
Po,,. - equivalent of 1 pack/day exposure to
tobacco smoke as source
over 25 years, gives 10% turnours in animals.
Radon - not good carcinogen in experimental
animals, needing much
higher dose than predicted from earlier radiation
source
studies - exposure requirement in dogs 13,000
working
level months.
Radon + smoke - smoking after radon dose increases
turnorigenic
response, smoke before radon greatly decreases
response
due to increased mucociliary clearance.
Po,,,, + non-specific lung proliferation inducers
(e.g. saline instillations) -
much increased response.
Mention was made of various studies designed to
seek agents which
protected against radiation-induced tumorigenesis
- Vitamin C, retinoids
and protease inhibitors.
Bond,J
Interactions with particulates - particular
reference to carbon black and
BaP.
Dosing with BaP on carbon black increases lung
covalent binding of BaP
as much as 10x compared with BaP alone. DNA
adducts increase over
air, but no difference in total between with and
without C particulate.
However, use of particulate does induce extra
btpes of adduct, also an
increase in metabolism. Lipid solution is
considered of more importance
than metabolism.
A different particulate effect is seen in the
human bronchus, where no
adducts form with bronchial tissue alone, but do
In the presence of
macrophages. C:)
CD
00
-Ij
C7%
ON
BATCo document for Province of BritiSh Columbia 28
October 1999


43
-A -
Gases and particles. Mention was made of an assay
of non-specific
irritants in lung tissue which uses hydroxyproline
incorporation into new
protein (collagen), reputed to be responsive and
useful.
C)
C:)
%-0
00
cr%
BATCo document for Province of British Columbia 28
October 1999


44
573 Sleight, S D and Rangga-Tabbu, C
Toxic effects of nitrosamines
1, 10 and 100 mg of NDELA/kg were injected Vp into
rats and compared
with other nitrosarnines used at equirnolar
concentrations. Effects on
target organs were examined after 24h in nasal and
liver tissues.
Differential effects on tissue response and
potency were observed using
NDELA, NPYR, NMP, DEN and NNN, which it is
hypothesized may be
significant in determining turnorigenic effects.
(It was difficult to rationalise
the specific effects claimed in the abstract with
those cited in the poster.)
4 ~b
CD
C=)
1.0
CO
C7%
CO
BATCo document for Province of BritiSh Columbia 28
October 1999


45
571 Hurtt, M et at
Methyl bromide and olfactory function
Exposure of rats to 200 ppm methyl bromide for 6
hours led to severe
destruction of the olfactory epithelium and loss
of olfactory function.
Recovery of function followed after 4-6 days
despite extensive morphol-
ogical damage remaining. A no-effect-level for
this of 90 ppm was
determined, compared with the current ACGIH
control level of 5 ppm.
C:)
C:)
%-D
CO
BATCo document for Province of BritiSh Columbia 28
October 1999


46
Agricultural Chemicals - Posters
702 McCarty, J D et al.
Lifespan feeding studies with Ethion in mice and
rats at levels up to 40
pprn produced no effects except for depression of
cholinesterase activity.
The no effect level was determined to be 4 ppm in
rats and 1.5 pprn in
mice.
699 Williamson, E G et al
Fenvalerate was significantly more toxic (acute)
when examined as the
formulated product Pydrin by comparison with the
technical grade
chemical in corn oil. The data suggest a
significant interaction between
the parts of the formulated product, which are
still under investigation.
705 Stott, W T et al
An inhalation study of technical grade
1,3-dichloropropene determined
ro-effect levels of 20 ppm and 5 ppm; in rats and
mice. At 60 pprn there
was an increased evidence of benign lung turnours
in mice.
405 Shane, 8 S et al
Monitoring of urinary mutagens as a means for
detecting exposure to
genotoxic agents was proposed in this paper.
Observations in green-
house workers identified an apparently clear
relationship between pres-
ence of urinary mutagens and exposure to genotoxic
pesticides. With
orthene (acephate), use of protective clothing
during spraying had a clear
and beneficial effect.
C)
CD
1.0
ca
_Ij
C:)
BATCo document for Province of British Columbia 28
October 1999


47
Vinyl Acetate
Three papers on this subject were presented by
staff from Hazleton UK: -
646 - a 2 yr study in drinking water; 658 - a 2 yr
inhalation study; and 969
- 2 generation reproduction study.
There were no significant pathological effects in
the feeding study and the
no-effect level was 1000 ppm. For inhalation, the
no-effect level was 50
ppm and an excess of nasal tumours occurred at 600
ppm.
The reproductive study produced marginal evidence
of a reduction in male
fertility at 5000 ppm vinyl chloride.
135 Lake, B G et al
Coumarin-hepatotixicity in rats. A biochemical
study from BIBRA sup-
ported by MAFF which has produced evidence for a
difference in
metabolism between coumarin and dihydrocournarin
which it is believed is
relevant to differences in hepatotoxicity. The
results are consistent with
coumarin 3,4-epoxide being the toxic intermediate.
CD
C:)
110
CO
BATCo document for Province of BritiSh Columbia 28
October 1999