TABLE 5

PREDICTED AND OBSERVED TIMES-TO-INCAPACITATION

_Time-to-incapacitation, minutes

7

PO AHFOO PH SADH OM THON MAOH OWNDNOOWOOAONONODULOMAP HPAL ONT POA HAA

EAI AD AN ED IN SE 909 9.69 0907 SECT OT ODE HE SH SLO LO LD LDL 1 HLA SHES OE COO TINE OM OLN OLN MOM INI C000

Predicted

from Eq.

co

SMO POME MANOHMHADDODAMOANANOADNOWONNOOODNOONAMOTFANMOADNE OHNNIAOMONAN
DODD PMO SF NN ADNAN AFOOTMNMRANODE OdAHAAANMNKRORADAROODANOAMDONOAAHAHODN ON
AN aed dea dd ANddidNddddiddididdtdndaNd Nadel Adana

Predicted
fr

AAR TAM OR AMANO ORNATE NEON DAOD OTN AF ADOMNODAOFADAM ATO TPMATOMCINO
SPOS ODA NH OLAS PLS A A SOLS AONE DOO OAN AOE DANDATAA COON ODAIADODN OAS
dq a a ANN ad do dadNddNddN NS NAN ddd

Predicted
from [HCN

| oannnodonaaninanoOORRNGAanniNanNrannodnnnnndnest+eNNENAGACNOonONe
a reee asad ara arasaatear an ar Sr rarer Mian B0 R00 20 OEE DE OE SE Oe Oe EO
AAA 09 0907 9 7 SST ST STS St ttt HLA LN OW OO OM AEE NAE HOMO HOMO OD OMOAHAAAACSS

17


to "walk" his front feet on the moving drum surface without
actually changing his location. Time-to-incapacitation was not
recorded until this "walking" motion ceased.

Exposures to CO alone. A plot of C vs t for a typical
experiment is provided as Figure 3; for this exposure: tj=11.9
min; integrated area from t=0 to t=t; is 29,048 ppm*min; average
CO concentration is 2,441 ppm (29048/11.9 = 2441).

The principal data from the 42 exposures to CO alone are
listed in Table 1. The average CO concentration (column 1) was
calculated from the integrated area (column 3) under the C vs t
curve, as described under MATERIALS AND METHODS; column 2 lists
the corresponding ty's, as observed experimentally.

Figure 4 is a scatter plot of tj vs tcoj1 for the 42
exposures conducted with CO alone. The equation for the curve
that was fit to these data is:

([CO]-225) * (ty-0.3) = 25,017. {Eq. 4}

A plot of this equation has been superimposed on the data points
in Figure 4. The toxicokinetic interpretation of the fitted
parameters in this equation is as follows: The value of the mean
effective dose (ED or Ko, see Eq. 1), for exposure to CO alone,
is 25,017 ppm*min; the minimal CO concentration that will

produce incapacitation (Cy) is 225 ppm; and the shortest exposure
time (t )) for which incapacitation can be produced at any
concentration is 0.3 min.

Exposures to HCN alone. Figure 5 shows the variation of
(HCN] as a function of exposure time for a typical experiment.

In this case, tj = 7.7 min, integrated area from t=0 to t=ty is
1,024 ppm*min, and average [HCN] is 133 ppm (1024/7.7 = 133).

The principal data from the 30 exposures to HCN alone are
listed in Table 2, where the column headings are analogous to
those for Table 1. Figure 6 is a scatter plot of ty vs [HCN] for
those specific exposures. The equation for the curve that was
fit to these data is:

({HCN) - 63) * ty = 564. {Eq. 5)

A plot of this equation has been superimposed on the data points
in Figure 6. In this case, an asymptotic value of zero for the
limiting time-to-incapacitation (t )=0) gave as good a fit to the
data as any nonzero value; therefore, we elected to use t)=0 in
this instance even though in other studies t . has had values up
to 0.1 min. The estimated values for the other toxicokinetic
parameters are: mean effective dose (ED or Kg)=564 ppm*min and
minimally effective concentration (Cg)=63 ppm HCN.

lgquare brackets signify concentration; [X] = concentration
of gas, X, in ppm.

11


14

PREDICTED Ti, MINUTES

Q — rn nm 4. -
Q 2 4 6 8 18 le

OBSERVED Ti, MINUTES

*
Figure 7. Observed vs predicted time-to-incapacitation for rats
exposed to CO-HCN mixtures

* Predicted t, was determined using equation 10.

21


and, although the contribution to lethality from CO alone might
predominate in some instances, in others it would be a case of
combined toxicity contributed from two or more individual com-
ponents. We felt that a candidate likely to make a significant
contribution to the overall toxicity, in addition to that due to
CO, would be HCN. The measurement of blood cyanide concentration
was therefore added to that for COHb in the standard protocol for
forensic toxicological analysis of autopsy specimens from victims
of aircraft accidents involving fire.

Our prediction concerning cyanide involvement was verified
when levels of HCN higher than normal were found in the blood of
victims of a DC-8 crash at Anchorage, Alaska, in which a post-
erash fire occurred (1). Several practical questions, however,
remained unanswered: What is the effective (incapacitating or
lethal) dose for each of these gases acting alone? How does one
add up their combined effects? Are their effects additive?
Synergistic? Antagonistic?

Subsequently, we established incapacitating and lethal
inhalation dose levels for CO and HCN and developed equations for
predicting time~-to-incapacitation (ty) and time-to-death (tg) for
rats exposed to the individual gases. We also established a
rationale for using experimental animal data to predict human
inhalation toxicity for CO and HCN (2). In some preliminary
experiments, we exposed rats to CO-HCN mixtures and found that
the sum of the two fractional effective doses (i.e., the quantity
of each gas that was inhaled divided by the quantity of that gas
required to produce incapacitation) was approximately 1.2, which
we felt probably represented experimental and biological vari-
ation about unity, or a simple additive effect (3).

Whether the effects of CO and HCN, when inhaled as a
mixture, are additive or synergistic has been the subject of some
research and considerable speculation over at least the past 60
years, since Hofer (4), in 1926, reported exposing cats to such
mixtures and measuring the exposure time required to produce
paralysis. A recent critique of 11 such studies was published
by Tsuchiya (5) and, although it is not a complete review of the
literature, it is interesting that the conclusions reached by the
individual authors were almost equally divided for and against a
synergistic combined effect. Tsuchiya attributed these differing
conclusions to one or more of the following deficiencies:

(a) improper use of the term synergism; (b) conclusions by the
authors that were inconsistent with the experimental data; or
(c) valid statistical treatments were not applied to the experi-
mental data.

Our continuing interest in this long-standing problem
prompted us, in 1985, to design a series of experiments that
would re-examine the question in sufficient detail to allow a
meaningful and useful interpretation of the results. This report
describes the design, execution, results, and the authors'
interpretation of the results of those experiments.


The logic for Eq. 7 is based on the following relationships:

(a) dose is proportional to toxic gas concentration;
(b) response time is inversely proportional to concentration;
(c) therefore ty 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 clearly illustrates that for most of
the 63 combined exposures the sum of the FED's is significantly
greater than unity (mean value: 1.23; std. dev.: 0.17; p<0.01
that the mean is equal to 1.00). This would suggest that one, or
both, of the component gases is contributing less to the combined
effect than one would predict from an exact summation of their
individual contributions; in other words, the combined effect is
less than exactly additive.

The data presented in the first three columns of Table 5 do
indicate, however, that for a large majority of the exposures (56
of 63) the response time (observed tj) produced by the
combination of gases is equal to, or less than, either of the
tj's predicted for one gas alone (mean tj from CO alone = 12.7
min, from HCN alone = 11.0 min, mean observed tj = 6.4 min). In
other words, the presence of the second gas did increase the
toxicity of the mixture beyond that attributable to the first gas
alone; and even in the seven cases for which the observed ty is
larger, the magnitude of the difference (0.2 to 0.6 min) is not
highly significant in light of the precision of the original
dose-response data.

The fourth column in Table 5 lists the tj calculated from
the relationship expressed by Eq. 7, i.e., the sum of the
reciprocal response times. If the effects of the two gases were
exactly additive, the values in column 4 should equal the
corresponding values in column 1; it is obvious that most of the
observed tj's are greater than the calculated (predicted) ones
(mean observed ty = 6.4 min; mean predicted tj = 5.1 min; linear
regression of observed on predicted gives a slope = 1.12,
intercept = 0.61, and a correlation of 0.91). Once again, this
can be interpreted as one, or both, of the gases contributing
less than its theoretical toxicity to that of the mixture.

At this stage of data evaluation, an obvious conclusion
would seem to be that the toxicity of an atmosphere containing
either of these two gases is increased by the simultaneous
presence of the other. The magnitude of the combined toxicity,
however, is less than would be predicted from an exact summation
of the individual toxicities. one question yet to be addressed
pertains to the magnitude of fractional contribution made by each
gas to the total effect.

Elucidation of degree of fractional contributions. As
previously stated, there are three functional mechanisms by which
the reduced toxicity of the combined gases might be explained.

We have evaluated the experimental data in the following manner

18


REFERENCES

National Transportation Safety Board. National
Transportation Safety Board Accident Report, Capitol
International Airways, Inc., DC-8-63F, N4909C,
Anchorage, Alaska, November 27, 1970. Report No.
NTSB-AAR-72-12, File No. 1-0025. Washington, DC:
National Transportation Safety Board, 1972.

Crane CR, Sanders DC, Endecott BR, Abbott JK,

Smith, PW. Inhalation Toxicology: I. Design of a
Small-Animal Test System. II. Determination of the
Relative Toxic Hazards of 75 Aircraft Cabin
Materials. Report No. FAA-AM-77-9. Washington, DC:
Federal Aviation Administration, Office of Aviation
Medicine, 1977.

Sanders DC, Crane CR, Smith PW, Abbott JK, Endecott
BR. ects of Exposure to Carbon Monoxide and
Hydrogen Cyanide. In: Preprints of the 1975 Annual
Scientific Meeting of the Aerospace Medical
Association. San Francisco, CA: Aerospace Medical
Association, 1975:137-138.

Hofer R. On the Effect of Gas Mixtures (in German).
Naunyn-Schmiedebergs Archiv fur Experimentelle
Pathologie und Pharmakologie 1926;111:183-205.

Tsuchiya Y. On the Unproved Synergism of the
Inhalation Toxicity of Fire Gas. J Fire Sciences
1986;4:346-354.

Scoggins MW. Ultraviolet Spectrophotometric
Determination of Cyanide Ion. Anal Chem 1972;
44(7):1294-6.

Pranitis PAF, Stolman A. Spectrophotometric Deter-
mination of Cyanide in Biological Materials. J
Forensic Sciences 1977;22:443-5.

Marquardt D. An Algorithm for Least Squares
Estimation of Nonlinear Parameters. J Soc Indust
Appl Math 1963;11(2):431-41.

24

¥U.S, Government Printing Office + 1888 - 617-000/03622


TABLE 6

OBSERVED AND PREDICTED TIMES-TO-INCAPACITATION

Time-to-incapacitation, minutes

10

Predicted
from E

{—_

Observed
tj

AAHONTSPOMONON OMINDAHATOOPOAMINAONO OMA WMNENTADNOOHAONADWVOOKO

AMANO MAMMA TESTO SHPO ITAL OL IG OCON

PTO LAADOAOOMAAAAM OOO NM ODANMA AMON ONACANAADAAM STE HNNONN DOHOMOODOND
AANA AN 1090909 0909 SH SS TS SH SR SH SH SHLD LDL LN OC OW DOOR EA AEKAE WMD DDODDDDDMDNONAANHAOCOO
dad

20



TABLE 3

DOSE-RESPONSE DATA FOR MIXTURES OF CO AND HCN

Gas Concentration,

ppm
co

Time-to-incapacitation,

minutes

HCN

WOMAN AADOWHOONOMOTOADHNATAMSAOCONONMDTONDAAOANMOIPTANMAAOAOD
ADMD DDD A DIANE NOOHOOMONNHNO MOOR NATE OAMOONDOGRANOMAMAOH SH
COPFIMMN OBOE RPORN STO SOTO ANDOR UMNO TOOMNIRMNEOMNNATANONW
AMANDA NAO IN FIN ANAM ANNAN AN ANN dN NINA Nd

RENADINOOCTOOPNOPPAAADAR MTN OMRON MOMAMNMARANRAM NON APHAOWOMAONMM OAM
DE AAANADAMNAMOOAR ADE DOODDNADO DAN MMAIMAAASAMNADAOHDOAADOMADAMAAMAAOOOD
AANAAARRAANN ddd Aa datdddedddtdidddddteididddddea deo dd aed ao ddda

SPFODDAD CH ODNDADANNAMOVOR RE DDADAMN AMON DNNOANAAAN AM SSH HOMO MOO HOMOONOND

VIVRE 7.09.09 09.09 ST SE ST SD tt PS SH SLA LOLA 19 (O10 WO SOLD LOTTE 00 60 0900 60 00 60 09 69 CD MAA AAS OS
d

i5


rotation, shock grid, and sampling timer for the GC were all
activated simultaneously. The rat was observed for any changes
in behavioral pattern, such as slowed response to shock,
gasping, collapse, convulsions, etc., and the time of onset and
duration of such occurrences were noted. The rat was considered
to be incapacitated when he no longer walked on the drum surface
and was carried onto, and remained on, the shock grid. The
elapsed time between insertion of the animal and this loss of the
ability to walk was recorded as ty After incapacitation, the
unconscious animal was removed from the chamber, weighed, and
sacrificed by immersion in a closed container filled with co.

Syringe samples for spectrophotometric analysis of HCN were
removed at approximately 1, 2, 3, 5, 7, and 10 minutes, then, if
necessary, at 3- to 4-min intervals until incapacitation was
observed. Chamber atmosphere samples were analyzed gas
chromatographically for CO and oxygen concentrations, with the
sample-loop timer programed to sample at 1 minute into the
exposure and at 1.8-min intervals thereafter.

The HCN dose/response relationship under these conditions
was determined by exposing 30 rats, individually, to mean HCN
concentrations of 75 to 273 ppm and measuring the time-to-
incapacitation while monitoring the HCN concentration in the
atmosphere. The dose/response relationship for CO, alone, was
determined in a similar fashion for 42 rats exposed to mean CO
concentrations that ranged from 1437 to 5922 ppm.

HCN and CO concentrations that would produce 5-, 10-, and

20-min tj's from the individual gases alone were combined for
the series of mixed-gas exposures, i.e., 5~min [CO] + 5~-min
[HCN], 5-min [CO] + 10-min [HCN], ..., 20-min [HCN] + 20-min
[CO]. Sixty-three rats were exposed individually to CO-HCN
mixtures using the same technique described for the single gas
exposures. HCN concentrations ranged from 84 to 215 ppm; CO
concentrations ranged from 1332 to 6385 ppm.

Data conversions and calculations.

Average gas concent. ion. For both the single-gas and the
mixed-gas experiments, the average concentration to which the
animals were exposed was calculated in the following manner:

The area under the "concentration vs exposure time" curve,
integrated from time=0 to time= =tis represents the (C*t;) product;
division of this product by t; yields the average effective
concentration to which the animal was exposed (C= Cty / tj).

Dose-response modeling for individual gases. For each set
of individual gas exposures, a scatter plot was constructed to
display the magnitude of ty as a function of exposure
concentration. A smooth curve, fit to these points, has the
shape of a rectangular hyperbola, with nonzero asymptotes:

(C-Cy) #(ty-ty) = Ko. {Eq. 1}


Figure 1, Animal exposure chamber

1.

Sensor, activates shock assembly when rat is
on grid

Shock grid

Gas deflector

Gas input port

Waste tray

Gas exhaust port
Rotating drum assembly

Latches for chamber cover


MATERIALS [D_ METHODS

Animals. Male albino rats of Sprague-Dawley origin were
obtained from Charles River Breeding Laboratories, Wilmington,
MA. They were ordered in a weight range of 100 to 120 g, were
inspected by a veterinarian on receipt, and were held in iso-
lation for 10 days prior to use. All were maintained for the
first 5 days on drinking water containing 1.5 g/L of sulfa-
thiazole, then on normal tap water for the remaining 5 days'
isolation.

Rats were fasted overnight before testing in order to
establish equivalent metabolic states; individual animal weights
were determined post-exposure.

Exposure chamber. The animal exposure chamber used for this
study was procured from Columbus Instruments (930 N. Hague Ave.,
Columbus, OH) and is illustrated in Figure 1. The assembly
consisted of a motor-driven drum and a shock grid mounted inside
a clear polymethylmethacrylate chamber (43.4 x 11.4 x 44.6 cm),
along with the associated electronic control devices. The
peripheral linear velocity of the drum was controlled at 6
cm/sec (0.134 mph) and the shock intensity was set at controller
position #5 (grid potential of 61 volts and maximum current of
0.61 milliamperes). As can be seen from Figure 1, if the subject
animal fails to maintain the selected walking speed he will be
carried back onto the shock platform.

Gas handling equipment. Carbon monoxide (research grade)
and breathing air were supplied as compressed gases by Big Three
Industries, Inc. (11426 Fairmont Parkway, La Porte, TX 77571).
Hydrogen cyanide was supplied by Matheson Gas Products (1920 West
Fairmont Parkway, La Porte, TX 77471) as a nominal 1500 parts per
million (ppm) mixture (volume/volume) in nitrogen.

The metered gases were mixed, as they were used, by passage
through a baffled cylindrical mixing chamber (3-cm dia x 23 cm)
before entering the animal exposure chamber. A gas sampling bulb
and a flowmeter connected in line between the mixing chamber and
the animal chamber allowed sampling (from the bulb sidearm) and
adjustment of the flow rate of the final gas mixture without
having to wait for equilibrium to be established in the large
chamber.

HCN flow rates were regulated manually with a double needle
valve assembly and monitored with a Matheson model 8116-0252 mass
flowmeter (Matheson Gas Products, Lyndhurst, NJ 07071). The pure
CO flow rate was regulated automatically with a Matheson model
8240 mass flow controller; air flow rates were controlled
manually by use of a simple, tank-mounted, two-stage pressure
regulator and a needle valve assembly.

The design of this gas delivery system is depicted
schematically in Figure 2.

Gas input to the chamber was through a port in the lower
front panel and exhaust was through a port in the rear panel.

3


TABLE 4

DERIVED DATA* FOR MIXTURES OF CO AND HCN

Fractional Effective

HCN

Dose

co.

HCN

Administered Dose

DOOM HOOP MANA OA AD MATH RADTPFAMATR AVN OM ONNOMMEOUANYNADAMNMNNODAN NOONAN
AAAAANDO OA MMOTAHMTANOTMANTMOAAMIOMAMAAS ADDAANAAIAMMINATOMM MAE

od 4 ddd dididdididididtdtdt ddd ddd dO ddd ddd ddd ddd

ROM ATYW AHO OOAN MNANTONNANOON TOODON TOW AYOAARMNMADMEHYAOMMOTHMAOVM MOAN
LIE AMO MYMPAADAOPAAAMMANONODAODHDON CONNOR NODMDHOON TOR AOMROYMAOTHAMDOOr

° dad d d d a d o

AOMPOTPINRMATHO ONAN ON RMRK FON DODON ODO TAADODONOONTOAMNMNYTONONNAVNO
PPI OON NOOR MIRMNDAPHFAADNOANANNNONNNOMMM TOPO PD PAPOMMOP POOP HPHOTATONIO
° a fo}

NOONE ONDAFRODOAIMANAAON MOON A ANANNOOWUHOOWOANTONOHUMTADONMOMNHNODO
ADANOVARKRANCMHAMARMAAPROAANATANATONDADDOANTRONDONOMNPTAMERADPTONOTHRM TAINAN
AN PAAMAN AND APA NAAT TOAD OM SETS SSH POSES IEMAMANTHAMNANDONNONT TOS

NE CWGANAHADOFHFHONON AHO ON DOMUONNNOODODHADOHOOMPOOCOMKYMONMOOOWTAOUHDOS
DALAARMDOTROAOMMMOFTAR TRAN DARAAT AR OANTATMNOCONOTMMOMOYMNAKNOIERAMMMATM
AT AOADTPNDONDMN DD AHO TANDDANADTOONOOADEOCNADNTOATOONAKHDODOMMNADATOADDAMAMO
DANOTFAMMOODHDODDOFOAMMAMOTINONOFEMERAMNONOOTNDOTDOTDHODNONOTOONATAIAMAS
ddidddddddd dd N AANNNN N Ad dd ddd dd ddd ddan AddNAAdNA Nd dda

POONANDOAODOAAANMOOON AN OAANMN AMON DADOIAANAA AN Sst HINO OD HOMO ONONOY

AAAAAN AAA AAT TS SH TH IAA COO CGONRERE NN 00900 HHODDGOOARAAACCS
ddd

See text for definitions of derived data.

*

16



The incoming gas flow was deflected upward by an internal 45°
deflector that was added to the system by the authors. Uniform
mixing inside the chamber was accomplished by running the motor-
driven drum while equilibrium was being established. The entire
chamber was located inside a fume hood through which the
exhausted gas mixture was vented.

Gas analysis. The chamber atmosphere was analyzed gas
chromatographically for CO and oxygen concentrations. The gas
chromatograph (GC) was equipped with 1/8-inch packed columns and
a thermistor detector. A gas sampling port was located in the
top of the exposure chamber, approximately 2 cm above the head of
the walking rat.

For the experiments with CO alone, 30-mL samples of the
chamber atmosphere were withdrawn manually into dry, plastic
syringes. These aliquots were then manually flushed through the
GC sample loop at a flow rate of 55 mL/min and injected from the
loop while maintaining this flow, so that the gas pressure-~and
thus the concentration--in the loop would match that produced by
the automatic pumping used later in the combined gas experiments.

For the combined gas studies, a continuous stream of the
chamber atmosphere was pumped (at 55 mL/min) from the port and
through the sample loop of the GC using a ceramic piston pump
(FMI model RRP; Fluid Metering, Inc., 48 Summit st., Oyster Bay,
NY 11771) with Saran tubing connections. Injections into the GC
were accomplished automatically by driving the sample loop
injector valve with an interval timer. Sampling interval was
limited to 1.8 min by the column retention time for Co.

Samples for HCN analysis were withdrawn manually from the
sampling port into acid-washed, all-glass hypodermic syringes--
syringes with metal ferrules on the tip must not be used. The
HCN was reacted, in the syringe, with the ammoniacal nickel
reagent described by Scoggins (6) and by Pranitis and Stolman
(7)# the concentration of the resulting tetracyanonickelate anion
complex was determined spectrophotometrically at 267 nm. Aqueous
standards for the spectrophotometric method were prepared daily
from reagent grade sodium cyanide (NaCN). The purity of the NacNn
was determined by titration with silver nitrate solution that had
been standardized previously against potassium chloride (primary
standard grade).

Test procedure. Before each test, flow rate from the
compressed air tank was adjusted to 1 L/min using the in line
flowmeter. The HCN and/or CO flow rates were then adjusted to
produce the concentration(s) desired at equilibrium, as
determined by the analysis of samples from the gas sampling bulb.
When the desired equilibrium concentration was achieved in the
chamber, the drum rotation was stopped and the chamber cover
latches were unfastened, but the cover was left on. The shock
intensity switch was set at position #5. At time zero, the
chamber cover was opened just enough to admit the rat, the rat
was inserted, the cover was closed and secured, and the drum

6


HCN is only about two-thirds as effective as when administered
alone.

The real significance of the result predicted from these
pieces of circumstantial evidence would be difficult to assess
from the data at hand. It also would be difficult to verify
experimentally except by first proposing the mechanism by which
the presence of CO reduced the toxic effect of HCN, and then
seeking experimental verification of the predictions based on
that hypothesis. The utility of the results from the present
study do not depend, however, on specific knowledge of the degree
to which each gas contributes to the combined effect, but on the
ability to predict ty as a function of the individual gas
concentrations.

Prediction of t; for mixtures of COQ and HCN. The
statistically-derived relationship presented as Eq. 9a can be
used to predict the response time that would result from a
mixture of known concentrations of the two gases, provided that
these concentrations are within those limits used in the original
experimental exposures. Re-naming the response variable from
1/ti(ebs) to 1/ti (predicted) and substituting the concentration-
dependent relationships for 1/ti(coy and 1/ti (Hon) (from Eqs. 4
and 5, respectively), Eq. 9a becomes:

1 + 0.68 = 1
0.3+(25,017/([CO}-225))  564/([HCN}-63) t 4 (pred) {Eq. 10}

Table 6 lists the observed ty's along with the ty's
predicted by Eq. 10 for each of the 63 exposures. Figure 7 is a
plot of the correspondence between these two sets of values, and
includes the least-squares regression line that was fit to the
data. The resultant equation is:

ty (pred) = 0.38 + 0.92 * ty (obs). {Eq. 11)

For this regression, the correlation coefficient is 0.92 and the
standard error of the estimate is also 0.92.

SUMMARY AND CONCLUSIONS

Rats were exposed, under controlled conditions, to selected
atmospheric concentrations of: (i) carbon monoxide in air; (ii)
hydrogen cyanide in air; and (iii) mixtures of carbon monoxide
plus hydrogen cyanide in air. For each animal, and for each
experimental condition, the elapsed exposure time (ty) required
to produce physical incapacitation was measured.

The response times for those experiments that utilized co
and HCN alone were plotted against exposure concentration and
equations were derived by nonlinear regression techniques that

22


FAA- Arm - 69-4

poo FAMIAMHESI4 INHALATION TOXICOLOGY: IX.

kat at Avion Mego Times-to-Incapacitation for Rats

89 ‘ Exposed to Carbon Monoxide Alone,

“ to Hydrogen Cyanide Alone, and to
Mixtures of Carbon Monoxide and

Hydrogen Cyanide

Charles R. Crane
Donald C. Sanders
Boyd R. Endecott

Civil Aeromedical Institute
Federal Aviation Administration
Oklahoma City, Oklahoma 73125

January 1989

Technical Report

This document is available to the public
at, . through the National Technical Information
“© * Service; Springfield, Virginia 22161.

US. oy fen Borys Pad
of Transportation
FAA Federal Aviation

Administration

89/04

Doc

E.R.A.U. LIBRARY


3888

2508 Pe 4

2088
1588

1888

CO CONCENTRATION, PPM

T1 = 11.8 min —

5aa

a 2 4 6 8 18 12 14 16
EXPOSURE TIME, MINUTES

Figure 3. CO concentration vs time, for typical experiment

50
45
40
35
30
25 x
20

TIME-TO-INCAP., MIN

15 ¥

a 2000 4028 $888 aaa
CO CONCENTRATION, PPM

Figure 4, Scatter plot of time-to~incapacitation vs CO concentration


Therefore, an equation of this form was derived empirically for
each data set, using a standard nonlinear regression algorithm as
a guideline (8), and identifying realistic values for the three
parameters: Cy, ty, and Ky. The biological interpretation of
these parameters is as follows:

Co is the minimum toxic gas concentration that will result
in incapacitation following a lengthy exposure;

to is the shortest tj that can be produced from an
overwhelming concentration;

Ky is an expression, in units of ppm*min, of the mean
"effective dose" (ED) of the specific toxic gas required
to produce the measured effect when administered alone.

e-response modeli °: e combined gas exposures.
The approach utilized by the authors to devise a mathematical
model for the combined effects of the two gases is based on the
concept of "fractional effective doses." This concept is
discussed in more detail in the RESULTS AND DISCUSSION section.
For each combined-gas experiment, an average gas concentration
was calculated for each of the two gases, using the technique
described for the single-gas exposures. Then, using the
rearranged dose-response equation {Eq. 1} derived for each gas,
the "administered dose" (AD) was calculated:

AD = K! = (C'-Co) *(ty'-to), {Eq. 2}
where:
ct is the average concentration of the specific gas
in the combined exposures;
ty! is the observed tj;

Co and tg are the derived constants for each gas from Eq. 1.

From the above data, a fractional effective dose (FED) was
calculated for each gas, in each exposure. The FED is defined as
the ratio of the administered dose to the effective dose, i.e.;

FED = AD/ED = K'/Ko. {Eq. 3}

RESULTS AND DISCUSSTON

Animal observations. During a typical test, the rat would
walk normally on the moving surface of the rotating drum after
only one contact with the shock grid. As the animal began to
respond to the toxic atmosphere, its walking rate slowed and more
frequent contact with the shock grid occurred, with the animal
occasionally jumping back and forth from the grid to the drum
surface. With continuing exposure, the rat often would allow the
rotation of the drum to bring his hindquarters into contact with
the grid and would cease to move his rear feet while continuing

8


HCN Mass
in Flowmeter
Nitrogen

oa

Air

Mass Flow
Controller

Carbon
Monoxide

UII]

Flow-
meter

Baffled
Mixing
Chamber

Sampling
Bulb

Exposure

Chamber

Figure 2, Diagram of gas delivery system

Vent


in an attempt to identify the most likely of those three
mechanisms.

The sum of the individual FED's (Eqs. 3 & 6) was observed to
be greater than unity (Table 4); however, if each FED were to
represent the real fractional contribution made by that gas to
the achievement of the observed effect (and incapacitation was
achieved), then the sum should equal 1.00. It occurred to us
that we could modify Eq. 6 by assigning a coefficient (a
weighting factor) to each of the FED's and setting this new sum
equal to 1.00:

a*FED(co) + b*FED(qcoN) = 1-00. (Eq. 8}
Statistically-derived values for the coefficients, a and b, could

then be obtained by doing a least-squares, linear regression
analysis on a rearranged form of Eq. 8:

FED(co) = 1/a - (b/a)*FED (Hen) - (Eq. 8a)

The least-square's best estimates for the coefficients are:
a= 1.05 and b = 0.57;
therefore, Eq. 8 could be rewritten as:
1.05*FED (co) + 0.57*FED (HCN) = 1.00. {Eq. 8b}

This statistically-derived result can be interpreted as
indicating that CO is contributing to the combined effect with
essentially the same potency that it would exert were it present

alone, while HCN is only 57% as effective in the presence of CO
as it would be alone.

A similar line of logic was then applied to forcing the sum
of the reciprocal ty's to equal the reciprocal of the observed
tj, see Eq. 7. The modified equation would then be:

a/ti(co) + b/ti(uen) = 1/ti(obs)- {Eq.9}
A multiple linear regression was performed on this set of data
using the algorithm that forces the constant term (or intercept)
to equal zero. The resultant least-square's estimates for the
coefficients are:

a = 1.00 and b = 0.68,

and Eq. 9 becomes:

1/ti(coy + 0-68/ti (Hen) = 1/ti(obs)- (Eq. 9a)

This result also suggests that the CO is contributing an
essentially undiminished potency to the combined effect while the

19


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

INTRODUCTION

The inhalation of toxic thermal decomposition products is
now recognized as the primary cause of death in most fire related
accidents. It is further recognized that even sublethal concen-
trations of these fire-generated gases may lead to psychophysical
incapacitation and thus prevent a victim's escape from the fire
environment. Of these gases, two that are likely to be produced
in quantities sufficient to pose a threat to life are carbon
monoxide (CO) and hydrogen cyanide (HCN). CO is produced from
all carbon-containing materials--and is produced in particularly
high concentrations when thermal decomposition occurs under
smoldering, low-oxygen conditions. HCN is a common thermal
decomposition product of any material that contains nitrogen
(wool, polyurethane, polyamides, acrylonitriles, etc.).

The Aviation Toxicology Laboratory of the Federal Aviation
Administration has been concerned since the mid-1960's that, in
the event of an inflight or postcrash fire, the diverse materials
present in aircraft cabin interiors could generate thermal
decomposition products of significant toxicity. Inhalation of
these toxic gases by passengers and crew who had survived any
attendant impact trauma could lead to their incapacitation and
subsequent failure to evacuate successfully.

In 1967 a research effort was initiated that was designed to
assist in the post-mortem identification of those fatalities,
from aircraft accidents involving fire, that were attributable
solely to smoke inhalation. This activity was placed in the
Forensic Toxicology Research Unit, under the direction of
Delbert J. Lacefield, and consisted of the post-mortem
measurement of the blood carboxyhemoglobin (COHb) concentration
in autopsy specimens from all victims of such accidents--for at
this time it was, and to a large degree still is, common practice
in the forensic medicine arena to describe death from smoke
inhalation as "CO poisoning." By 1970 it had become obvious that
many such victims, with no evidence of physical trauma, were
reported to have blood COHb levels that were consistent with the
diagnosis of death due to CO poisoning~-and, thus, to "smoke
inhalation." In a significant number of cases, however, the COHb
saturation level was reported by Lacefield as considerably less
than 50 percent and it was, therefore, questionable to assign the
cause of death as CO poisoning alone (or even as smoke
inhalation) on the basis of evidence at hand.

The authors were aware that smoke from burning materials
contains a multitude of individual thermal decomposition products


HCN CONCENTRATION, PPM

TIME-TO-INCAP., MIN

158

125

188

75

58

25

Ti = 7.7 min --——->

Figure 5,

5@
45
48
35
38
25
2a
15
18

2 4 6 8 18
EXPOSURE TIME, MINUTES

HCN concentration vs time, for typical experiment

Figure 6.

58 188 158 208 25a 30a 358 480
HCN CONCENTRATION, PPM

Scatter plot of time-to-incapacitation vs HCN concentration

12


Exposures to CO-HCN mixtures. The times-to-incapacitation

that resulted from the 63 individual exposures of a single rat to
a defined mixture of CO and HCN are listed in Table 3, in order
of increasing tj.

The average concentrations of CO and HCN to which the
animals were exposed (C', Eq. 2) were calculated as they were for
the single gas exposures, and from this data the "administered
dose" (AD, or K'), as defined by Eq. 2, was derived. The
fractional effective dose (FED), as defined by Eq. 3, was
calculated for each of the two gases and for each exposure. A
listing of these calculated values, as well as the sum of the two
FED's for each exposure, is presented in Table 4.

Once the administered dose is known for each gas, we also
can calculate the predicted ty that should result if an animal
were exposed to that concentration of that gas alone. This is
accomplished, for both CO and HCN, using equations 4 and 5
respectively, and the results are presented in Table 5.

Evaluation of CO-HCN interaction. In the viewpoint of the

authors, there are at least two techniques available to us for
determining whether or not the toxic effects of the two gases are
additive. The first approach is based on the simple concept that
the sum of the fractional effective doses, due to each gas alone,
should equal unity if their combined effects are exactly
additive. The second relationship is that the sum of the
reciprocal ty's, predicted for each gas separately, should equal
the reciprocal of the observed ty, for the combined gases, if the
toxic effects are exactly additive. The equations describing
these two relationships are:

FED(co) + FED(Hcn) = 1.0 {Eq. 6}
and

W/ticco) + VWticneny = 1/ti(ops)- (Eq. 7}

The logic of the FED concept can be illustrated by the
following. If one administers to a subject, by injection, one-
half of a lethal dose of chemical A followed immediately (or
simultaneously) by an additional one-half lethal dose of chemical
A, then by definition lethality should result. The end-result
should be the same if the two fractional doses were three-fourths
plus one-fourth, or two-thirds plus one-third, etc. This
conclusion is obvious because the two doses administered are of
exactly the same chemical and one would, therefore, expect their
combined actions to be exactly additive. If, on the other hand,
one administers two dissimilar chemicals and their combined
effects achieve the defined result, then one could conclude that
their actions are exactly additive provided the sum of their
FED's is unity. If their combined effects are synergistic, one
would expect the sum of the FED's to be considerably less than
unity; if the sum is significantly greater than unity, then their
combined effects must be antagonistic or "less than additive"
(including the possibility of no interaction whatsoever).

14


Technical Report Document

¥. Report Ne. 2) Government Accession No 3. Ree ta Cotelog No
DOT/FAA/AM~89/04
4. Title and Subtitle 5. Report Dere

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

7 awe) Charles Re Crane, Donald C. Sanders,

and Boyd R. Endecott

Performing Orgenizenven Code

AAM-613

Perlorming Orgonizatien Repart No.

DOT/FAA/AM-89/04

9. Pestorming Orgen ranon Name and Address
FAA Civil Aeromedical Institute
P.O, Box 25082

Oklahoma City, OK 73125

Work Unit No. (TRAIS)

Contract or Grant No.

12. Sponsoring Agency Name and Address
Office of Aviation Medicine
Federal Aviation Administration
800 Independence Avenue SW

Type of Report and Pariod Covered

TECHNICAL REPORT

Jan. 1986 to Nov. 1986

Sponsoring Agency Code

Washington, D.C. 20591

15. Supplementary Notes

This work was performed under task AM-B-86-TOX-53,

16. Abstract

Laboratory rats were exposed to experimental atmospheres that contained (a) carbon
monoxide in air, (b) hydrogen cyanide in air, and (c) mixtures of CO and HCN in air.
The toxic potency of each of the three types of environments was evaluated toxico-
kinetically by measurement of time-to-incapacitation as a function of the toxic gas
concentrations. Regression equations were derived that describe those relationships
for exposure to CO or HCN alone. Analysis of the data from the combined-gas
exposures, and comparison of that data with the results obtained from the exposures
to each gas alone, lead to the conclusion that the toxicity of the combination is
definitely greater than would be produced by either gas alone. There was, however,
no evidence for a synergistic action--in which the effect of the combination would
have been greater than that predicted from the sum of the two individual effects.
An empirical equation was derived that allows calculation of a predicted
time-to-incapacitation for any combination of CO and HCN concentrations that are
within the ranges utilized in the experimental exposures.

17. Key Words 78.
Combustion toxicology; smoke; carbon
monoxide; hydrogen cyanide; time-to-
incapacitation; aircraft cabin fire.

idution Stotement
This document is available to the public

through the National Technical Informa-
tion Service, Springfield, VA 22161.

19. Security Classil. (of this report) 20. Security Classsf. (of this poge) 21. No. of Pages

27

22. Price

Unclassified Unclassified

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized



described those relationships mathematically. The resulting
equations, for time-to-incapacitation, are:

ty = 0.3 + 25,017/([C] - 225), for CO, and

ty = 564/((C]-63), for HCN,

where tj is in minutes and [C] is the concentration, in ppm, of
the toxic gas in the atmosphere. The response times obtained
from exposures to mixtures of the two gases were then analyzed
with respect to the response time that would have been predicted
from the action of each individual gas acting alone at the
concentration utilized in the mixture.

Our conclusion from this analysis is that the toxic potency
of a mixture of CO and HCN is greater than can be ascribed to the
same concentrations of either gas by itself--at least for the
concentration ranges utilized in this investigation. The data
do not support, however, the hypothesis of synergism--that is,
that the combined effect would be greater than that predicted by
summing the two individual effects. The mechanistic hypothesis
that is supported by the data is that the toxic potencies of
these two gases are fractionally (or incompletely) additive when
they are present in the same atmosphere at concentrations that
fall within the range included in this investigation. With
emphasis on this important caveat, we offer an equation that may

be of value in estimating the time-to-incapacitation produced by
mixtures of CO and HCN.

1
ti (pred) = ( 1 ( 0.68 )
0.3+(25,017/([CO]-225))/ + \564/([HCN]-63)/.

23


NOTICE

This document is disseminated under the sponsorship of the
Department of Transportation in the interest of information
exchange. The United States Government assumes no liability
for its contents or use thereof.

The animals used for this experiment were lawfully acquired
and treated in accordance with the "Guide for the Care and Use
of Laboratory Animal Res6urces," National Research Council,
DHEW Publication No. (NIH) 74-23,


Pred. *
Tir
min

(C*Ti),
ppm*min

TABLE 1

DOSE~RESPONSE DATA FOR CARBON MONOXIDE ALONE

DOIPPOAN SOO DAOUOMR MOON YNRNADONTIMAANTHOROONN
DOMDOKN AA KNMNANANHAHOODADAARDDONOMNNNNNNNM NII
ANd datasets

CMM OMM OOADATOVATOON NO TOM ANTOMODOSONNATNANONTIND
HODDOUN FOMOODPOAMHDDOIHNAMOONOTONR TNdOOTNG
CRAPPTONOAFADAOHDONNATOORANTOTODOSAHANOR RY HOGM
WAIN AOMNANTOOONDONMNORTOMNARARMONONOTNRRROTHOOd
ANNA MMNAN NA AAANA MANO NAM MM ANAAAM ANAM MNANAAMAMA

AAMT OM PFOAHOPFIHDOOTNMOTFADMNTPOWVONDNAN WOdHOOMANO

WOM FOCODAMNTATANDAODSCOCHUOHDON NUNN TNUNNNNN SOM
ANNANAddAddididddded Aaoiddd

ROOK AMO MATAR ANDOAMAMDOMPONNONI MN AAIMOASHOAN
AR COLFHTODNOANO TIM MO ATONAL MANATOMPOROPIANTION
PIOOOO OOK AAMIM SF PFOROAAHDOANTONOTHAMMMTHPIOON OM
AAA GAA AANA NN AIAN OR 69 09.09 09.09 SHLD LOD LOD LOD LOD LO LOLI

0.3+(25,017/([CO]-225)).
10

*predicted ty



TABLE 2

DOSE-RESPONSE DATA FOR HYDROGEN CYANIDE ALONE

Pred.*
(HCN), Tj, (C*T3), Tj,
ppm min ppm*min min
75 21.9 1642 47.0
83 20.8 1726 28.2
89 21.4 1905 21.7
94 20.2 1903 18.2
95 17.6 1672 17.6
96 22.6 2170 17.1
98 19.8 1950 16.1
99 10.6 1049 15.7
100 21.1 2116 15.2
100 16.3 1630 15.2
107 11.2 1198 12.8
112 8.2 918 11.5
112 13.4 1501 11.5
113 10.2 1153 11.3
119 10.6 1261 10.1
128 7.3 934 8.7
129 13.0 1677 8.5
133 7.7 1024 8.1
140 8.1 1134 7.3
156 6.9 1076 6.1
158 6.7 1059 5.9
174 5.7 992 5.1
180 4.6 828 4.8
187 5.1 954 4.5
216 3.4 734 3.7
218 3.4 741 3.6
221 3.9 862 3.6
232 2.9 673 3.3
245 3.0 735 3.1
273 2.8 764 2.7

*Predicted ty = 564/([HCN]-63).

13