mean ratios of the observed t,'s dividea by the t; for each gas
in the mixture Were 1.15 and 1.12 for CO and for acrolein

Observed in all 10 cases, this slight difference between observed
t and the Predicted individual gas t;'s was not statistically
Significant at the P o5 level using Student's t test.

Selection of combined gas concentrations for the preceding
Comparisons was limited to the concentration ranges used in
defining the original dose~response curves for the individual
gases, i.e., 1119-6524 Ppm for CO ang 580-41550 ppm for acrolein,
However, one suggested mechanism for the decrease in apparent
toxicity of co in the presence of even Small amounts of acrolein
is that the acrolein irritates the mucous membranes, thereby

fraction. Therefore, we plotted the ratios of the observed t;'s
to the calculated t's for the co alone (since the co
concentration Was within the limits of our dose-response curve)
against the mean acrolein concentrations for each of the 15 low-
acrolein exposures. This Plot is shown in Figure 5,

18

|



rhe
"

combined gas exposures should equal the sum of the reciprocals
for the calculated t,'s for each gas.

1/t,ccoy + 1/t,cacroteiny = 1/t ; (observed) (Eq. 4)

TABLE 3

DOSE-RESPONSE DATA FOR MIXTURES OF CO AND ACROLEIN

T, (min) (CO), ppm (Acrolein), ppm
4.4 3202 14967
4.7 3052 13421
5.5 1318 11310
5.8 1464 12445
6.0 5518 11283
6.7 5843 5028
6.7 6050 12416
6.8 6152 5297
7.0 2877 12344
7.4 3161 4476
8.3 5573 4761
9.1 3535 4823
9.4 3683 4922
9.7 5834 2136
10.6 1547 5196
11.0 3354 4282
11.1 2998 4174
11.4 1474 4897
12.6 5421 1830
15.0 3227 1281
17.2 3010 960
18.0 3070 1839
18.5 1321 1213
20.3 1480 1476
28.1 1463 1382

The second concept is that of the fractional effective dose.
When equations 2 and 3 are rearranged as equation 1, the
individual effective doses (K,) for CO and acrolein are 22839 and
35192 (ppm*min) respectively. From the observed t,'s and
calculated mean gas concentrations (C) for rats exposed to the
gas mixtures, we calculated the fraction of this "effective dose"
that each animal inhaled at t,. Thus the fractional effective

12



_10.

11.

12.

13.

14,

15.

16.

Iwanoff N. Experimentelle Studien uber den Einfluss Technish
und Hygienisch Wichtiger Gase und Dampfe auf den Organismus;
Teil XVI, XVII, XVIII: Uber einige Praktisch Wichtige
Aldehyde (Formaldehyd, Acetaldehyd, Akrolein). Archiv fur
Hygiene. 1910; 73:307-40,

Jacobs MB. The analytical chemistry of industrial poisons,
hazards, and solvents, 2nd ed. New York: Interscience
Publishers, Inc.; 1949:401-9, 678-9.

Kaplan HL, Grand AF, Rogers WR, Switzer WG, Hartzell GE.

A research study of the assessment of escape impairment by !
irritant combustion gases in postcrash aircraft fires.

Washington, Dc: Department of Transportation/Federal

Aviation Administration;1984; FAA publication no.

FAA~CT-84-16. Available from: National Technical

Information Service, Springfield, VA 22161. Order

#ADA146484.

Lewin L. Uber die Giftwirkungen des Akrolein; ein Beitrag
zur Toxikologie der Aldehyde. Archiv fur experimentelle
Pathologie und Pharmakologie. 1900; 43:351-66.

Lynch RD. On the non-existence of synergism between inhaled
hydrogen cyanide and carbon monoxide. Fire Research Note

No. 1035, Fire Research Station, Borehamwood, Hertfordshire,
England, May 1975.

Marquardt D. An algorithm for least squares estimation of
nonlinear parameters. J Soc Indust Appl Math. 1963;
11(2) 2431-41.

McNamara BP. Estimates of the toxicity of hydrocyanic acid
vapors in man. Report No. EB-TR~76023. Aberdeen Proving
Ground, MD: Department of the Army/Headquarters, Edgewood
Arsenal, 1976. Available from: National Technical
Information Service, Springfield, VA 22161.

Order #ADA028501.

Morikawa T. Acrolein, formaldehyde, and volatile fatty
acids from smoldering combustion. J Combustion Toxicol.
1976; 3:135-50.

Moss RH, Jackson CF, Seiberlich J. Toxicity of carbon

monoxide and hydrogen cyanide gas mixtures. Arch Ind Health
1951; 4:53-64.

Murphy SD, Davis HV, Zaratzian VL. Biochemical effects in
rats from irritating air contaminants. Toxicol Appl
Pharmacol. 1964; 6:520-8.

22



for the first 4 days, then normal tap water for the remaining 4
days' isolation. The rats were fasted overnight before testing
to establish equivalent metabolic states; each animal was weighed
immediately prior to use.

Exposure chamber design. The animal exposure chamber
utilized in this study has been previously described (3); its
design is detailed in Figure 1. It differed from the chambers
used in earlier studies in that its design allowed the insertion
of the test animal directly into an established test atmosphere,
while retaining the rotating cage assembly for the determination
of t,. Chamber construction was of polymethylmethacrylate (PMMA)
with internal dimensions 50.8 cm long by 26.2 cm wide by 50.6 cm
high. The two-compartment cylindrical rotating cage, 40.6 cm in
diameter by 25.0 cm wide, had a plastic mesh floor (perimeter)
and a perforated divider. The cage was suspended across the
width of the chamber by a central axle attached to the perforated
divider; the outer chamber walls functioned as end walls for the
cage. Chamber access ports (10 cm by 10 cm), equipped with
gaskets, were installed in each side of the chamber at the level
of the rotating cage floor, to allow rapid insertion or removal
of the test animals. The rotating cage was driven by a 4-rpm
geared motor (Dayton model 3M098), providing a linear velocity
(circumferential) of 8.5 cm/s.

Gases. CO (99.5%, CP grade) and breathing air were obtained
from Big Three Industries, Inc., La Porte, TX. Acrolein was
purchased from the Aldrich Chemical Co., Inc., Milwaukee, WI, as
a 97% solution containing approximately 3% water and 200 ppm
hydroquinone to inhibit polymer formation. This material was
purified by fractional distillation and refrigerated; the
acrolein was brought to room temperature and centrifuged to
remove any polymer (disacryl) before use for test atmosphere
generation or preparation of gas chromatographic standards.

Test atmosphere generation. The amount of acrolein required
for a given experiment was calculated from the desired
atmospheric concentration and the enclosed chamber volume (67.35
L). A side door was removed from the dry, empty chamber, and the
calculated volume of freshly centrifuged liquid acrolein was
introduced by pipet into a 10-cm glass Petri dish centered on the
chamber floor. The door was replaced and the fans were turned on
to promote evaporation and atmospheric mixing; equilibrium was
considered to be complete when all liquid acrolein had
evaporated, a period of 15 to 45 min, depending on the quantity
added.

CO atmospheres were produced by direct injection of pure CO
into the chamber using a gas syringe; equilibration of the co
with chamber air was complete within 1 min, although time
requirements for test sampling and animal handling usually
permitted a 5- to 10-min equilibration time before animal
insertion.


20
| +
18 4
174
1.64
185
144
135
S124
2115
ye 4 -4
Sos ty/t,(CO) = 1.22 + (7.2 x 10°" * Acrolein, ppm)
vos 4
9 0.7 4
5 oe 7 (CO) = 3280 ppm (s.d. = 180)
3 04-4 t, (CO) = 8.6 min (s.d. = 0.49)
S034
0.2 4
ot 4
0.0 t t T T T

° 200 400 600

Acrolein concentration, ppm

Ratio

Figure 5. Observed t/t, (CO) at low acrolein concentrations

The least-squares regression indicates a slow, somewhat
erratic increase in the observed t,/t,(CO) ratio with increasing
acrolein concentration. Perhaps more significant is the increase
above the mean t,(CO) for the series (8.6 min, s.d.=0.49,
c.v.=5.7%) at the lowest acrolein concentrations (y-intercept is
1.2*t,(CO). It would appear that low levels of acrolein
effectively increased the t, above that normally expected from
the CO concentration.

SUMMARY AND CONCLUSIONS

Laboratory rats were exposed individually to selected
atmospheric concentrations of CO in air, acrolein in air, and
mixtures of CO and acrolein in air. The exposure time required
to produce physical incapacitation in each test animal was
measured for each experimental condition.

19


REFERENCES

Crane CR. The prediction of human incapacitation by the
combustion products carbon monoxide and hydrogen cyanide
using a small animal test protocol. In: Preprints of

the 1975 Annual Meeting of the Aerospace Medical Association.
San Francisco, CA: Aerosp Med Ass'n. 1979;151-2.

Crane CR, Sanders DC, Endecott BR, Abbott JK. Inhalation
toxicology: Iv. Times to incapacitation and death for rats
exposed continuously to atmospheric hydrogen chloride gas.
Washington, DC: Department of Transportation/Federal
Aviation Administration;1985; FAA publication no.
FAA-AM-85~4. Available from: National Technical Information
Service, Springfield, VA 22161. Order #ADA157400.

Crane CR, Sanders DC, Endecott BR, Abbott JK. Inhalation
toxicology: VII. Times to incapacitation and death for

rats exposed continuously to atmospheric acrolein vapor.
Washington, DC: Department of Transportation/Federal
Aviation Administration;1986; FAA publication no.
FAA-AM-86-5. Available from: National Technical Information
Service, Springfield, VA 22161. Order #169666.

Crane CR, Sanders DC, Endecott BR. Inhalation toxicology:
IX. Times-to-incapacitation for rats exposed to carbon
monoxide alone, to hydrogen cyanide alone, and to mixtures
of carbon monoxide and hydrogen cyanide. Washington, Dc:
Department of Transportation/Federal Aviation Administration;
1989; FAA publication no. FAA-AM-89-4. Available from:
National Technical Information Service, Springfield, VA
22161. Order #ADA208195.

Einhorn IN. Physio-chemical study of smoke emission by
aircraft interior materials. Part I. Physiological and
toxicological aspects of smoke during fire exposure.
Washington, Dc: Department of Transportation/Federal
Aviation Administration;1973; FAA publication no.
FAA-RD-73-50. Available from: National Technical Information
Service, Springfield, VA 22161. Order #AD763602.

Goldbaum LR, Ramirez RG, Absalon KB. What is the Mechanism

of Carbon Monoxide Toxicity? Aviat Space Environ Med. 1975;
46:1289-91,.

21


longer t, than the CO. However, when acrolein was present in a
concentration that would produce a shorter t, than the CO, the
observed t, was governed by the acrolein concentration, with
: little effect from the CO. A summary of these comparisons is
presented in Table 6.

TABLE 6

PAIRED STUDIES ~~ COMPARISON OF PREDICTED TIMES-TO-INCAPACITATION
(t,'s) FOR INDIVIDUAL GASES WITH OBSERVED t,'s FOR THE i
CARBON MONOXIDE-ACROLEIN MIXTURES |

*Predicted t,'s (min) Observed t,'s_for |
Acrolein Carbon’ Monoxide Co} |
EQUAL
TOXICITY 5.1 5.4 6.0
PAIRS 4.9 5.0 6.7
9.1 8.9 7.4
8.6 8.0 9.1
, 8.5 7.7 9.4
9.4 8.4 11.0
t 9.5 9.3 11.1
mat 21.6 21.9 18.5
19.1 19.3 20.3
wt 19.6 19.5 28.1
\f
, UNEQUAL 8.4 5.2 6.7
i TOXICITY 8.1 5.0 6.8
t PAIRS 8.7 5.4 8.3
8.6 19.4 11.4
8.2 18.4 10.6
4.4 8.8 4.4
4.7 9.2 4.7
4.9 9.7 7.0 l-
5.1 22.0 5.5
4.8 19.5 5.8
15.0 5.2 9.7
16.7 5.5 12.6 j
20.9 8.7 15.0 .
24.8 9.3 17.2
16.7 9.1 18.0 -“
*Predicted t,'s for acrolein and CO were calculated from the
concentrations of each gas in the combined exposures, using
equations 3 (for acrolein) and 2 (for CO) respectively.
16



guideline (12),
For cO, a nonlinear regression on the data set for the 25
individual exposures resulted in values of -714, -1.8, ang 46350

t;) to an overwhelming concentration Of the gas, and
K, is an expression in Ppmtmin units of the mean
"effective dose" of a toxic gas requireg to produce
he measured effect (incapacitation .

Earlier work by the authors (4) indicated that 225 Ppm was a
Practical estimate for Cc, and a 1.1-min estimate for t, gave a
good fit for the higher co Concentrations on the concentration-
versus~t, curve, Using these estimates, nonlinear regression

0 Of 22839, The resulting equation (Eq. 2,
below) is Plotted over the data Points (see Table 1) from which
it is deriveq (see Figure 2).

t forco = 4.4 +(22839/(¢9 ~ 225)) (Eq. 2)

sug

18 * 5 = l10+ (22839/(co - 225))

14 Equation 2

to-Incapacitation, min

Time-
ones ae

ef
”
.
a
o

Figure 2, Mean carbon monoxide concentrations vs time~to-incapacitation

ee


CO-acrolein mixed atmospheres were prepared by the same
techniques, except that the CO was added during the last 10 min
of the acrolein equilibration period. In all cases, pre-exposure
concentrations were increased by approximately 7% to allow for
the expected concentration loss during rat insertion.

Carbon _m i an is. CO was analyzed by gas
chromatography, using a Carle model 8000 gas chromatograph (GC)
equipped with a thermistor detector. The analytical columns were
1/8" stainless steel, a 3-ft column packed with 40/60-mesh silica
gel connected in series to a 6-ft column packed with 40/60-mesh
molecular sieve 5A. The carrier gas was helium; column
temperature was 86°C. Standards were prepared by making serial
dilutions of tank CO with air in a 100-mL gas syringe. Sample
injection was accomplished by operating a solenoid-activated gas
sampling valve while manually injecting the syringe sample
through the sample loop at 55 mL/min.

Chamber atmosphere samples were withdrawn through a septum
in the chamber access port (from a point within 2-in of the rat's
normal head position) using 60-mL plastic syringes. These
aliquots were then manually flushed through the sample loop at
the same 55-mL/min flow rate and injected from the loop while
maintaining this flow, so that the gas pressure--and thus the
concentration--in the loop would match the injection condition of
the standards. CO concentrations were calculated from the peak
heights of the chamber aliquots using a least-squares regression
equation relating peak height and concentration of the gas
standards.

Acrolein analysis. Acrolein was also analyzed by gas
chromatography, using a Perkin-Elmer Sigma 2000 GC equipped with
a flame ionization detector. A tandem-connected quartz capillary
column was utilized, consisting of a 25-m by 0.25-mm i.d. section
coated with Carbowax 20M connected to a 20-m by 0.32-mm i.d.
section coated with methyl silicone. Helium was used as the
carrier gas at a linear velocity of 15 cm/sec; oven temperature
was 100°C.

Standards for gas chromatographic analysis were prepared by
injecting known weights of redistilled acrolein into Saran gas
bags containing 1000 mL of dry air, then making syringe dilutions
of the "bag" standards after equilibration was complete. In
application, a Saran gas bag (with septum) was flushed with room
air and evacuated using 3 consecutive fill/evacuate cycles.

After the last evacuation, the bag was injected with exactly 1000
mL of tank breathing air with a 1000-mL gas syringe.

Approximately 40 wL of redistilled acrolein (at room
temperature) was drawn into a 50-yL Hamilton syringe, the needle
was inserted into a small piece of septum to prevent leakage, and
the entire assembly was weighed on a microbalance to the nearest
0.01 mg. After injecting the acrolein into the prepared Saran
gas bag, the syringe assembly was reweighed and the weight of the
injected acrolein was determined by difference. The acrolein

5


6.
7.
8.
9.

10.

ll.
12.

—4—4
5 Pio
+t ite}
6 T
DS -
TT igasl
E BIN2
_S ——!|

Figure 1, Animal exposure chamber

Mixing fan assembly, consisting of Dayton model 2M033
motor, 1/15 hp, 5000 rpm, 120 VAC, 60 Hz, fitted with a
4-bladed, 7-cm dia nylon fan,

Ventilation holes, 12-mm dia, cut through center divider
of rotating cage,

Petri dish, 9-cm dia.

Exposure chamber walls constructed of ‘sin (12-mm) thick
polymethylmethacrylate (PMMA).

Rotating cage assembly, center divider and outer rim
constructed from ~in (6-mm) PMMA,

Gas sampling ports sealed with serum vial stoppers.

Chamber access port for animal insertion and removal.
Thumbscrew fasteners,

Polyethylene mesh cover for rotating cage; mesh openings
are approximately 7-mm square.

Center divider and Support for rotating cage, constructed*
from k-inch (6mm) thick PMMA,

Cage drive motor; Dayton model 3M098, 4 rpm, 120 VAC, 60 Hz.
Cross supports for chamber rims and plastic mesh perimeter.



When the predicted t,'s were calculated by Eq. 4, the sum of
the reciprocal response times, all of the predicted t,'s were
shorter than the observed values. Linear regression of observed
on predicted t,;'s gave the equation;

tjtobserved) = -0.25 + 2.26*t (predicted)
with a correlation coefficient of 0.89.

At this stage, we attempted to identify the relative
magnitudes of the toxicities for the two gases in the combined
gas atmospheres. Although the sum of the individual FED's was
always greater than unity, we reasoned that since incapacitation
was achieved, that we might assign a weighting factor to each FED
and set up a new sum equal to 1.

a*FED(co) + b*FED¢acrotein) = 1 (Eq. 6)

Rearranging equation 6 into the form of a straight line (y=art
bx), we obtained

FEDcacrolein) = 1/b - (a/b) *FED¢co) (Eq. 6a) |
By performing a least-squares, linear regression analysis on the
data arranged as in equation 6a, we determined best estimates for
the coefficients a and b of 0.17 and 0.74 respectively.

0.17*FED(coy + 0.74*FED(acroteiny = 1 (Eq. 6b)

The same logic was applied to forcing the sum of the
reciprocal t,'s to equal the reciprocal of the observed t; \
(Eq. 4) by assigning a weighting coefficient to each of the
individual predicted t; reciprocals. The modified equation was:
a*l/tj(coy + b¥1/t;tacroleiny = 1/t, cobserved) (Eq. 7)

A multiple linear regression was performed on the data using the
algorithm that forces the constant term (or intercept) to equal
zero. The least-squares estimates for the coefficients were 0.23
and 0.74 for a and b respectively.

0.23%1/t,(coy + 0.74%1/t,Cacroleiny = 1*1/t (observed) (Eq. 7a)

17


sampling times was necessary for combined CO-acrolein exposures
because the samples for the different analyses were removed from
the same sampling port for uniformity.

During the exposure, rat behavior such as eye-blinking,
grooming, gasping, hyperactivity, and convulsions were noted and
recorded along with the time of observation. Time-to-
incapacitation was recorded as the elapsed time between insertion
and the time at which the rat could no longer perform the
coordinated act of walking in the rotating cage (i.e., when
sliding and tumbling began). After t,, a final chamber sample
was removed and analyzed to provide a complete time-concentration
record for the exposure period. Cage rotation was then stopped,
and the rat was removed and sacrificed by insertion into a closed
container of CO.

The acrolein dose/response relationship for these conditions
was determined by exposing 31 rats, individually, to selected
acrolein concentrations from 580 to 41550 ppm and measuring the
t, while monitoring the acrolein concentration in the chamber
atmosphere. The dose/response relationship for CO alone, under
the static atmosphere conditions necessitated by the CO-acrolein
combined exposures, was determined in a similar fashion for 25
rats exposed to CO concentrations from 1119 to 6524 ppm.

Acrolein and CO concentrations calculated to produce 5~-,
10-, and 20-min t,'s from each of the individual gases alone were
combined for a series of mixed gas exposures, i.e., 5-min (CO) +
5-min (acrolein), 5-min (CO) + 10-min (acrolein),...20-min (CO) +
20-min (acrolein). In addition, 15 rats were exposed to a 10-min
t, level of CO combined with very low levels (18 to 580 ppm) of
acrolein to study the irritant effect at subtoxic levels. Forty
rats were exposed, individually, to the CO-acrolein mixtures,
using the technique previously described for the single gas
exposures. CO concentrations ranged from 1318 to 6152 ppm, and
acrolein concentrations ranged from 18 to 14967 ppm.

RESULTS AND DISCUSSION

Data conversions and calculations. For all experiments, the
area under the concentration-versus-exposure-time curve, for each
gas, was integrated from time = 0 (i.e., at animal insertion) to
t, (incapacitation) ; division of the concentration*time product
by t, yielded the average gas concentration to which the animal
was exposed (i.e., C = C#t,/t;).

For each set of single gas exposures, a scatter plot was
constructed displaying t,; as a function of the mean exposure
concentration for the gas. A smooth curve, fitted to these
points, has the shape of a rectangular hyperbola with nonzero
asymptotes:

(c - C,)*(t; - t,) =, (Eq. 1)

An equation of that form was empirically derived for each data
set, using a standard nonlinear regression algorithm as a

7


17.

18.

19.

20

21.

22.

23.

24.

25.

26.

Pattle RE, Cullumbine H. Toxicity of some atmospheric
pollutants. Brit Med J. 1956; 2:913-6.

Pitt BR, Radford EP, Gurtner GH, Traystman RJ. Interaction
of carbon monoxide and cyanide on cerebral circulation and
metabolism. Arch Environ Health. 1979; 34:354-9.

Purser DA, Berrill KR. Effects of carbon monoxide on
behavior in monkeys in relation to human fire hazard.
Arch Environ Health. 1983; 38:308-15.

Salem H, Cullumbine H. Inhalation toxicities of some
aldehydes. Toxicol Appl Pharmacol. 1960; 2:183-7.

Sanders DC, Crane CR, Smith PW, Abbott JK, Endecott BR.
Effects of exposure to carbon monoxide and hydrogen cyanide.
In: Preprints of the 1975 Annual Meeting of the Aerospace
Medical Association. San Francisco, CA: Aerosp Med

Ass'n. 1975:137-8.

Skog E. A toxicological investigation of lower aliphatic
aldehydes: 1. Toxicity of formaldehyde, acetaldehyde,
propionaldehyde, and butyraldehyde; as well as of acrolein
and crotonaldehyde. Acta Pharmacol. 1950; 6:299-318.

Stewart RD. The effect of carbon monoxide on man.
JFF/Combustion Toxicol. 1974; 1:167-76.

Syracuse Research Corp., Center for Chemical Hazard
Assessment, NY. Information Profiles on Potential
Occupational Hazards: I. Single Chemicals, Acrolein.
Contract No. PHS-NIOSH-210-78-0019, (NTIS PB81-147951),
Final Report, Dec. 1979.

Tsuchiya Y. On the unproved synergism of the inhalation
toxicity of fire gas. J Fire Sci. 1986; 4:346-54.

United States Dept. of Commerce News. FP 78-7:105-7,
Apr 12, 1978.

23

*U.S, Government Printing Office: 1991-561-020/20597


E.R.A.U. LIBRARY

Federal Aviation Administration
Oklahoma City, OK 73125

December 1990

ent is available to the public trough
nal Technical Information Service,
pied, Virgina 22161.



40 |
|
33 4 i
H

& 34

a !

€

g 77

3 te 2.15 + (35192/(Acrolein + 595))

§ 24

zd Equation 3

a is-

§

c

"104

°

s

a s4

f

- t) r T . {

7
o - 20 40
Acrolein, ppm (thousands)

Figure 3. Mean acrolein concentration vs time-to-incapacitation

TABLE 2
DOSE-RESPONSE DATA FOR ACROLEIN

t;, min (Acrolein) , ppm t,, min (Acrolein) ,ppm
2.8 41550 7.7 5870
2.8 35010 10.0 5280
2.9 22590 10.2 3686
3.8 18170 12.9 3033
3.8 15060 13.2 2490
3.9 20710 13.5 4240
4.2 10990 14.1 1940
4.4 16720 19.7 1220
4.9 10060 19.8 1260
5.1 10540 21.4 1075
5.8 8505 21.6 1281
6.0 9341 21.8 900
6.4 6595 27.0 580
7.3 8630 27.3 1028
7.4 5150 34.0 580

36.5 690

10



TABLE 1

DOSE-RESPONSE DATA FOR CARBON MONOXIDE

t;, min (CO), ppm t,;, min (CO), ppm
4.6 6229 11.4 2954
4.7 6524 11.9 2893
5.3 6143 12.9 2700
5.4 5666 14.6 1974
5.5 5036 15.6 1345
5.8 5207 15.7 2606
5.8 5161 16.4 2240
6.8 4626 16.9 1696
8.2 3698 17.1 1879
9.8 2511 20.1 1380

10.4 3129 21.1 1297
11.4 2240 24.3 1234
25.3 1119

For acrolein (Acr), nonlinear regression for the 31
individual exposures produced values of 2.15, 35192, and -595 for
t,, K, and C, respectively. The resulting equation (Eq. 3) is
plotted in Figure 3 over the data points from which it is
derived; individual values are shown in Table 2 in order of
decreasing acrolein concentration.

tj for acrolein = 2.15 + (35192/(Acr + 595)) (Eq.3)

Although this equation describes the data, the negative
value for C, does not describe the toxicological minimum "no
effect" concentration. As suggested by the authors in the
earlier acrolein study (3), the fact that the model equation,

t,=t, + K/(C - ¢,)

could not be fit to the acrolein data by using a positive value
for C,, may indicate that the form of the equation is
inappropriate for describing the toxicokinetics of acrolein.

By relating t, to the square root of the effective
concentration, C - C,, and estimating values for t, and c, at 0.1
min and 300 ppm respectively, nonlinear regression resulfed in a


concentration in the bag was calculated at the ambient
temperature and barometric pressure, and a standard curve was
prepared by injecting syringe dilutions of the "bag" standard
into the gas sampling loop of the chromatograph. Unknown
acrolein concentrations were calculated from the peak heights of
chamber samples using a least~squares regression equation
relating peak height and concentration of the gas standards. The
accuracy of the GC technique was verified originally by analyzing
duplicate standard and test gas samples using a second,
independent spectrophotometric technique (3). The differences in
results obtained by the two methods never exceeded 5 percent.

The actual retention time for an acrolein sample in this
system was about 5.5 min, with a slightly longer time being
required for the recorder trace to return to baseline. Ina
static exposure system such as ours, the gas concentration slowly
decreases after animal insertion due to inhalation and to
adsorption on the chamber walls, animal fur, and solution in
expelled body fluids. We felt that more frequent sampling would
be desirable to adequately describe the area under the time-
versus-concentration curve for the individual animal exposures.
In this simple air-CO-acrolein atmosphere, the flame ionization
detector responded only to the acrolein with no injection
"artifact." This characteristic enabled us to inject samples at
4-min intervals and still allow the recorder to return to
baseline between samples; no difference in peak height was found
between samples injected in this manner and identical samples
that were allowed to return to baseline before the next injection
(about 8 min). Thus, we were able to schedule chamber sampling
at 0.5, 3, 7, 11, 15, 19, 23, 27, and 31 min. A vacuum line was
attached to the gas sample loop between injections to flush the
loop with room air. An obvious caveat to be noted is that this
type of sampling must be restricted to this, or similar simple
systems, where only a single component produces a response from
the selected detector; additional components should be carefully
checked for possible interference.

Chamber atmosphere samples were withdrawn through a septum
in the chamber access port into a 20-mL gas syringe and
immediately transferred to the sample loop of the GC. The
sampling syringe was flushed with room air 3 times between
samples.

Animal exposure procedure. When the test atmosphere had
reached equilibrium, a chamber air sample was withdrawn and
analyzed for CO and/or acrolein; the concentration was then
adjusted and resampled, if necessary, to reach the planned
exposure level. The cage rotation and fan motors were turned
off, and a weighed, fasted rat was rapidly inserted through the
chamber access port. The port was immediately resealed, the
exposure timer was activated, and the cage and fan motors were
restarted.

Sampling of the chamber atmosphere for CO began at about 0.5
min and continued at 1 to 3-min intervals. Some staggering of

6



t; = 1.10 + (22839/(¢ - 225)) for co, ana
t; = 2.15 4+ (35192/(c + 595)) for acrolein,

where t,; is in minutes and C is the toxic gas concentration in
Pparts-per-million by volume.

The response times obtained from exposures to mixtures of co

predicted t,'s for either gas in the mixture. Thus our
analytical data indicates that the toxic potency of a mixture of
CO and acrolein -- within the concentration ranges used —- was
less than or equal to that which can be ascribed to either gas by
itself.

The data do not Support the hypothesis of synergism, since
the sums of the paired FED's are all greater than unity. An
argument for an antagonistic effect is supported by the increase
in the observed t, over that predicted for CO in cases where a
“less than equipotent" concentration of acrolein is present. An
argument can also be made for "no interaction" on the basis of

Although the mechanism is not defined by this limited
investigation, we conclude that evidence does exist for an
"antagonistic-like" effect when acrolein is present in

in most fire conditions. The effect is Probably not antagonistic
in the sense that acrolein decreases the specific toxicity of co,
but that it increases the time required to inhale an effective
dose of co by decreasing the animal's ninute-respiratory volume
(MRV). Whether this decrease in MRV is a voluntary reaction or
an involuntary neural reflex is not known. For concentrations
within the ranges used in this study, we offer the following
multiple linear regression equation, which may be useful for
estimating time-to-incapacitation for rats exposed to atmospheric

1
0.23 } [ 0.74 )
1.10+(22839/([CO}-225))] + 2.15+(35191/([Acr]+595) )

t, =

20



is probably more familiar to the public as the acrid, tear-
producing odor associated with the overheating of animal fats and
oils. Structurally, it is a 3-carbon, unsaturated aldehyde with
the formula CH,=CHCHO. It is a suspected thermal decomposition
product of certain materials used in aircraft cabin interiors and
has been identified in 91% of 120 building fires monitored in a
1978 study by the Harvard School of Public Health (26).
Firefighters, in particular, have recognized the irritant and
toxic dangers of acrolein, particularly in low temperature (300-
400°C), smoldering thermal conditions involving polyethylene,
polypropylene, vinylon, and cellulose containing materials (14).

Considerable confusion exists in the literature concerning
the levels of acrolein likely to be incapacitating or lethal to
humans. Predicted (10-20 ppm) levels appear conservative (8,24)
when compared to experimental values for various animal species
(7,20), and are apparently based on discomfort indices instead of
actual physical incapacitation. Our earlier study (3) indicated
that approximately 4200 ppm of acrolein was necessary to produce
incapacitation in the laboratory rat in 10 minutes, and Kaplan
(9) found that baboons could successfully escape from an exposure
chamber after exposure to 2780 ppm for 5 minutes.

This study evolved from a need to better understand the
relative toxic contribution of individual combustion gases to the
overall toxicity of a mixture. No aircraft fire produces only a
single combustion product; therefore, if we are ever to predict
the probable physiological effect of a gas mixture, we must first
obtain an understanding of how the mixture components interact to
produce their effect on the experimental subject. Do two (or
more) components react in such a way as to be synergistic,
antagonistic, or are their toxic actions completely independent
of each other? There has also been speculation that irritant
gases, at low concentrations, might cause shallower breathing
with a lowered minute respiratory volume, thereby exerting a
“protective” effect against other non-irritant systemic toxicants
such as CO, resulting in a longer time-to-incapacitation (t,)
than would be expected from the CO concentration alone.

Our earlier work with CO, HCN, HCl, acrolein, and CO-HCN
mixtures (2,3,4,21) utilized a rotating cage assembly inside an
animal exposure chamber to measure t, for rats exposed to these
combustion gases. Similar procedures were used in this study to
relate t; to gas concentration for rats exposed to CO and
acrolein individually, and to CO-acrolein mixtures. Gas
concentrations in the mixtures ranged from 1318 to 6152 ppm for
co, and from 18 to 14967 ppm for acrolein.

MATERIALS AND METHODS

Animals. Male albino rats of Sprague-Dawley origin were
obtained from Charles River Breeding Laboratories, Wilmington,
MA, in a weight range of 100 to 120 g. The animals were
maintained on drinking water containing 1.5 g/L of sulfathiazole

2


dose (FED) of each gas inhaled from the mixture was equal to

FED = ((C ~ C,)*(t, - t,)]/K,

(Eq. 5)

where C is the mean concentration for the individual gas in the

mixture and t, is the observed time-to-incapacitation.

A list of

the calculated values for FED(CO), FED(acrolein), and the sum of
the FED's for each exposure is presented in Table 4.

TABLE 4

FRACTIONAL EFFECTIVE DOSES FROM EXPOSURES TO
CO-ACROLEIN MIXTURES”

FED(CO) FED(ACROLEIN)
0.430 0.995
0.446 1.016
0.211 1.133
0.255 1.352
1.136 1.299
1.377 0.727
1.428 1.682
1.479 0.779
0.685 1.783
0.810 0.756
1.686 0.936
1.159 1.070
1.257 1.137
2.112 0.586
0.550 1.390
1.356 1.226
1.214 1.213
0.563 1.444
2.616 0.720
1.827 0.685
1.963 0.665
2.105 1.096
0.835 0.840
1.055 1.068
1.464 1.458

FED(SUM)
1.425

1.462
1.344
1.607
2.435
2.104
3.110
2.258
2.468
1.566
2.622
2.229
2.394
2.698
1.940
2.582
2.427
2.007
3.336
2.512
2.628
3.201
1.675
2.123
2.922

“see text for definitions of Fractional

Effective Doses.

13


Logically, since the effective dose for a specific gas is
equal to K, (Eq.1), and the fraction of that effective dose
inhaled from the mixture at t, is described in Eq. 5, the sum of
the fractional effective doses for each of the gases in the
mixture should equal 1 if the combined toxic actions are exactly
additive. For a synergistic effect, the sum of the individual
FED's would, therefore, be less than 1. For an FED sum
significantly greater than 1, the possibility exists for an
antagonistic effect (i.e., that the presence of one gas decreases
the toxic effect of the other). Another possibility is that the
combined effects are "less than additive" because of differing
rates of action for each of the gases at the sub-incapacitation
dose levels attained at the observed incapacitation. A third
possibility is simply that the toxic mechanisms for each gas are
so different that each acts independently on the test animal and
no interaction occurs.

The logic for Eq. 4 is based on the following relationships:

(a) Dose is proportional to the toxic gas concentration;

(b) Response time is inversely proportional to gas
concentration;

(c) Therefore, response time (t,;) is inversely
proportional to dose.

As a consequence, if the combined dose effect is related to the
sum of the individual doses, then the combined response times
should be calculated as reciprocals. !
Examination of Table 4 indicates that for all of the 25
combined exposures, sums of the paired FED's are all considerably
greater than unity (mean FED sum = 2.28, std. dev. = 0.56, 95%
confidence intervals on the mean = 2.05, 2.51). This would
suggest that the combined effects are much less than additive.
Table 5 indicates that for all but two of the combined
exposures, the observed t,'s were greater than or equal to the
shorter of the predicted t,'s for the individual gases. When
rats were exposed to concentrations of CO and acrolein selected
to produce equivalent t,'s, the observed incapacitation time was
not statistically different from the predicted t,;'s for the
individual gases at the Po; level. For example, the mean
predicted t,'s for 5 such exposures produced the following
values.

Predicted t,(co) =
Predicted t,¢ecroleiny = 9
Observed t,(co + acroleiny = 9.6

8.5 min (s.d.=0.65)
-O min (s.d.=0.45)
min (s.d.=1.53)

When t,(CO) was shorter than t,(acrolein), the observed t, was

longer than t,(CO). Conversely, when t,(CO) was longer than

t,(acrolein), the observed t; was only slightly longer than the
predicted t, for acrolein alone. This could be interpreted as
indicating that acrolein exerts a "protective" effect when it is

present in mixtures at levels that would produce, by itself, a

ie

15


INHALATION TOXICOLOGY: xX. TIMES TO INCAPACITATION FOR RATS
EXPOSED CONTINUOUSLY TO CARBON MONOXIDE, ACROLEIN, AND TO CARBON
MONOXIDE~ACROLEIN MIXTURES

INTRODUCTION

The development of fire in an otherwise survivable aircraft
accident greatly decreases the passengers' chances for survival,
both from direct fire effects and from the loss of capacity for
escape from the fire environment caused by breathing the toxic
components of smoke. Disorientation can occur, not only due to
the direct visual obscuration caused by smoke, but also because
of the lachrymatory effects of irritant gases within the smoke.
Concurrently, certain systemic toxicants, such as carbon monoxide
(CO) and hydrogen cyanide (HCN), are being inhaled that will
eventually result in physical incapacitation. Death, either from
continued inhalation of toxic gases or from thermal effects, is
the likely sequel.

While dose-response relationships for single gas exposures
have been defined for some of the major toxic components of smoke
(1,13,19,23) and for a few of the irritant gases
(2,3,7,10,16,17,20,22), less information exists pertaining to the
combined effects of these gases on the intact mammalian organism
(11,15,18,21,25). With present technology, it is not feasible to
reconstruct, or even to define, all of the complex mixtures of
combustion products that occur in aircraft fires. It is
possible, however, to determine the reaction of laboratory
animals to simple, defined mixtures of pure gases to better
understand the combined effects of these gases in the fire
environment. This study was designed to investigate the combined
effects of CO, a systemic toxicant, and acrolein, a toxic,
unsaturated aldehyde noted for its extreme irritant properties.

co is a colorless, odorless gas produced by the incomplete
combustion of carbon-containing materials, particularly under
smoldering conditions where insufficient oxygen is available to
allow further oxidation to carbon dioxide. The ubiquitous
distribution of such materials (such as cotton, wood, hydrocarbon
fuels, and most plastics) virtually assures that some CO will be
present in any aircraft fire. The classical mechanism for CO
toxicity is the rapid combination of CO with the hemoglobin of
the red blood cells, thereby reducing the oxygen-carrying
capacity of the blood (23). Other workers (6) have presented
evidence indicating that the more probable toxic action is one of
co on cellular respiration, where dissolved CO (in plasma)
crosses into the tissues and competes with oxygen for cytochrome
a, in the mitochondria. Whatever the actual mechanism(s),
carboxyhemoglobin levels of about 50% and 70% are generally
associated with incapacitation and death, respectively, in humans
(5,8).

Acrolein, the other toxic gas component in this study, was
briefly used as a chemical warfare agent in World War I (8), but

1



Technical Report Documentation Page

1, Report No.

DOT/FAA/AM-90/15

2. Goverment Accession No.

3. Recipient's Catalog Now

4, Title end Subtitle

INHALATION TOXICOLOGY: X. TIMES TO INCAPACITATION
FOR RATS EXPOSED CONTINUOUSLY TO CARBON MONOXIDE,
ACROLEIN, AND TO CARBON MONOXIDE-ACROLEIN MIXTURES

5. Report Dote
December 1990

6. Performing Orgenizatian Code

7 author’) caries R. Crane, Donald C. Sanders

and Boyd R. Endecott

- Performing Organization Report No.

9. Parferming Organizetion Neme ond Address

FAA Civil Aeromedical Institute
P. 0. Box 25082
Oklahoma City, OK 73125

10. Work Unit No. (TRAIS)

V1. Contract or Grant No.

12, Sponsoring Agency Nome and Address

Office of Aviation Medicine
Federal Aviation Administration
800 Independence Ave., SW
Washington, D.C. 20591

V3, Type of Report and Period Covered

14.” Sponsoring Agency Code

15. Supplementary Notes

This work was performed under task AM-B-87-TOX-55.

16. Abstroct

of toxic gas concentrations.

in the experimental exposures.

Laboratory rats were exposed to experimental atmospheres of (a) carbon monoxide
(CO) in air, (b) acrolein in air, and (c) to mixtures of CO and acrolein in air.
The toxic potency of each of the three types of environments was evaluated
toxicokinetically by measurement of time-to-incapacitation (t,) as a function
Regression equations were derived that relate

the observed t,'s to the concentrations of individual gases.

Comparison of the combined-gas-exposure data with results obtained from the
individual gas exposures indicated that there was no evidence of synergistic
action, i.e., that the effect of the combination was never greater than that
predicted by the sum of the two individual gas effects.
an inhibitory or antagonistic effect of undefined mechanism when acrolein was

present in the mixture at concentrations of lesser toxic potency than that of CO.

Evidence did exist for

An empirical equation was derived that allows the calculation of a predicted t
for combinations of CO and acrolein concentrations within the ranges utilized

17, Key Words 7 1
Inhalation” toxicology; ‘carbon monoxide;
acrolein; 4¥ritant gas;~€0-acrolein
toxicity; time-to-incapacitation;

animal exposure systems. VA 22161.

18. Distribution Stotement
Document is available to the public
through the National Technical
Information Service, Springfield,

¥9. Security Closail, (ol this report)

Unclassified

20. Security Cloasif. (of this page)
Unclassified

2. No, of Poges | 22. Price

26

Form DOT F 1700.7 (8-72)

Reproduction of completed page authorized



value of 589 for K, with a residual sum of squares (RSSQ) of 190.
This represents a slightly poorer "goodness of fit" than the
RSSQ value of 145 obtained for Eq. 3. The mathematical
relationship is expressed as,

t; acrotein = 0.1 + (589/(Acr - 300)°*) (Eq. 3a)
and the graphical relationship of Eq. 3a to its parent data is
depicted in Fig. 4. Reasonable predictions of response time for
specific acrolein concentrations can be made from either Eq. 3 or
3a within the concentration limits from which they are derived.

ty = 0.1 + (589/(Acrolein - 300)°°>)
Equation 3a

Time-to-Incapacitation, min
8
1

° T T t T
Acrolein, ppm (thousands)

Figure 4, Mean acrolein concentration vs time-to-incapacitation

Times-to-incapacitation resulting from 25 individual rat
exposures to defined mixtures of CO and acrolein are listed in
Table 3 with the average concentrations for each gas to which the
rat was exposed. The average concentrations were calculated as
for the single gas exposure, i.e., average concentration =
(fe dt)/t;. Two techniques were utilized to determine whether or
not the toxic effects of the two gases were additive. First, for
an additive effect, the reciprocal of the observed t, for the

11


oy

From the average gas concentrations in the mixtures, we
calculated the predicted t, that would result if the animal were
exposed to that concentration of each gas alone, using equations :
2 and 3. These predicted t,;'s are compared to the observed t, /

for each CO-acrolein exposure in Table 5.

TABLE 5

PREDICTED AND OBSERVED TIMES-TO-INCAPACITATION

Time-to-Incapacitation, minutes

Observed t, Predicted Predicted Predicted from
from [CO] from [Acrolein] Combined Gases
(Eq. 2) (Eq. 3) (Eq. page 20)
4.4 8.8 4.4 5.2
4.7 9.2 4.7 5.4
5.5 22.0 5.1 6.4
5.8 19.5 4.8 6.1
6.0 5.4 5.1 5.3
6.7 5.2 8.4 7.5
6.7 5.0 4.9 5.0
6.8 5.0 8.1 7.3
7.0 9.7 4.9 5.7
7.4 8.9 9.1 9.3
8.3 5.4 8.7 7.8
9.1 8.0 8.6 8.7
9.4 747 8.5 8.6
9.7 5.2 15.0 10.7
10.6 18.4 8.2 9.8
11.0 8.4 9.4 9.4
11.1 9.3 9.5 9.8
11.4 19.4 8.6 10.2
12.6 5.5 16.7 11.6
15.0 8.7 20.9 16.2
17.2 9.3 24.8 18.3
18.0 9.1 16.7 14.3
18.5 21.9 21.6 22.4
20.3 19.3 19.1 19.8
28.1 19.5 19.6 20.5

14