Long-term, low-level exposure of guinea pigs and marmosets to 
sarin vapor in air: lowest observable effect level 

Toxicology and Applied Pharmacology 189 (2003) 170-179 15jun03

Herman P.M. Van Helden,* Henk C. Trap, John P. Oostdijk, Willem C. Kuijpers,
Jan P. Langenberg, and Hendrik P. Benschop

Division of Chemical and Biological Protection, TNO Prins Maurits Laboratory, P.O. Box 45, 2280 AA Rijswijk, The Netherlands
Received 16 December 2002; accepted 15 January 2003

Realistic scenarios for low-level exposure to nerve agents will often involve exposures over several hours to extremely low doses of agent. In order to expose animals to the lowest controllable concentrations of agent and to increase exposure times until a lowest observable effect level (LOEL) becomes measurable, a validated system was developed for exposing conscious animals to 0.05-1.0.rg/m³ (8-160 ppt) of sarin and other nerve agents. Based on cold trapping of sarin from the exposure air, the concentration could be measured semicontinuously, at 4-min time intervals by means of gas chromatography. We found that the LOEL upon a 5-h whole body exposure of guinea pigs and marmosets to sarin vapor corresponds with the measurement of an internal dose by means of fluoride-induced regeneration of sarin from phosphylated binding sites in plasma, mostly BuChE. For guinea pigs the LOEL was observed at Ct = 0.010 ± 0.002 mg/min/m³, whereas a Ct of 0.04 ± 0.01 mg/min/m³ was established for the LOEL in marmosets. These levels are several orders of magnitude lower than those based on classical measurement of depressed cholinesterase activities. At low exposure levels of guinea pigs and marmosets (<1 µg/m3), a reasonable linearity was observed between exposure dose and internal dose. The data were addressed in the light of the recently recommended occupational exposure limits to sarin for workers without respiratory protection, which suggests that the exposure limits should be reconsidered if the slightest inhibition of cholinesterases should be prevented.

* Corresponding author. Fax: +31-15-284-3963. E-mail address: helden@pml.tno.nl (H.P.M. Van Helden).

Keywords: O-Isopropyl methylphosphonofluoridate; Sarin; Nerve agent; Respiratory exposure; Lowest observable effect level (LOEL); Guinea pigs; Marmosets; Fluoride-induced reactivation; Internal dose; Occupational exposure limits; Worker population limit (WPL); Short-term exposure limit (STEL)

Previous work on low-level exposure to chemical war-fare (CW) agents pertained to occupational exposure during planned destruction of these agents (McNamara and Leitnaker, 1971; see also Benschop et al., 1998). However, various developments, especially the terrorist attack with sarin in the metro of Tokyo (Croddy, 1995), lead to the notion that the effects of low-level exposure to CW agents on personnel become increasingly important under actual conditions of military or terrorist attacks with CW agents. For example, (1) small amounts of agent may penetrate through the closures and unnoticed slight damage of protective clothing or gas masks, (2) imperfections during donning and doffing procedures of protective gear will have the same effect, (3) personnel performing duty in collectively protected areas may be exposed to small amounts of agent due to entry and exit procedures and residual contamination of entering personnel, (4) both offgassing and physical contact with decontaminated material such as painted surfaces and protective clothing may contribute to low-level exposure, and (5) exposure of personnel due to a downwind transport of agent over long distances from contaminated areas, for example, due to destruction of enemy stockpiles, which was suggested as a possible contributing factor to the Gulf War Syndrome (Ember, 1996). Evidently, several of these scenarios may involve low-level exposures over several hours. For this reason we chose to perform our exposure experiments over a 5-h period, which is within the range of a working day. Many attempts have been made to define various degrees of low-level exposure to nerve agents (Solani and Romano, 2001). Rather than adhering to one of these definitions, our approach was to expose animals to the lowest controllable concentrations of agent and to increase exposure times until effects become discernible. The possibility that military personnel in a conflict situation or first responders and medical personnel in case of a terrorist attack may be low-level exposed to CW agents makes it necessary to ascertain that exposure has or has not taken place based on detection and reliable diagnosis/dosimetry of trace exposure. Moreover, it should be established at which vapor concentrations in air minimal systemic effects will become observable, but also at which concentrations these effects start to have adverse effects on performance (Van Helden et al., 2001).

For several reasons we have chosen sarin as the target compound for the study of effects of CW agents during low-level exposure: (1) several potential adversaries have stockpiled this agent for possible use in chemical warfare; (2) it has been claimed that US soldiers may have been exposed to sarin during or in the aftermath of the Gulf War when rockets filled with sarin were destroyed at Khamisiyah, Iraq (Ember, 1996); and (3) sarin has been used in terrorist attacks by the AUM Shinriyko sect in Matsumoto (1994) and Tokyo (1995) (Croddy, 1995; Polhuijs et al., 1997).

The ongoing controversy and discussions whether soldiers were low-level exposed to chemical warfare agents such as sarin during the Gulf War show clearly that drastic improvement with regard to sensitivity and reliability are needed for diagnosis/internal dosimetry of exposure to CW agents in general and to nerve agents in particular (for a review see Noort et al., 2002). We therefore developed a method to establish the internal dose of sarin based on the release of the inhibitor phosphyl moiety from butyrylcholinesterase (BuChE) and possibly other binding sites in plasma with fluoride ions followed by GC analysis of re-constituted sarin (Polhuijs et al., 1997). It is estimated that 0.01% inhibition of BuChE can be measured in this way, which is an improvement by approximately three orders of magnitude over previous methodology based on measurement of residual blood cholinesterase (ChE) activity.

We consider the measurement of the internal dose as essential for the determination of exposure levels (Ct) that correspond with the lowest observable effect level (LOEL), i.e., the exposure level at which sarin starts to penetrate into the systemic circulation. For these studies we selected guinea pigs and marmoset monkeys since an extensive basis of toxicokinetic data for nerve agents is available in the first-mentioned species whereas primates are more similar to humans with regard to binding sites for nerve agents (Benschop and De Jong, 2001).

In the present study (1) methods were developed and validated in order to expose guinea pigs and marmosets to extremely low concentrations of sarin (0.05-1.0 µg/m³,8-160 ppt) with on-line semi continuous analysis of the exposure concentrations of sarin and (2) the LOEL of exposure was determined, i.e., the Ct for exposure to sarin (t < 5 h) at which an internal dose becomes measurable in blood. Such measurements also provide preliminary data on the relationship between exposure dose and internal dose.

Warning. In view of its extreme toxicity and volatility, sarin should only be handled in specialized and government-approved laboratories where trained medical personnel are continuously present.

Animals. Male Dunkin-Hartley albino guinea pigs (Harlan, NL) with a body weight of 500-600 g were used. Male and female marmosets (Callithrix jacchus) with a body weight between 350 and 420 g were provided by Harlan, UK. Housing and care of the animals was according to standard operating procedures. Protocols for the animal experiments were approved by the TNO Committee on Animal Care and Use.

Equipment for generating, analyzing, and exposing guinea pigs and marmosets to sarin. See Results for a description of the equipment.

Fluoride-induced reactivation of sarin-inhibited BuChE in plasma. Heparinized blood, taken from the guinea pigs and marmosets via the carotid cannula, was stored on melting ice (<5 min). After the blood volume was determined, the sample was centrifuged for 4 min at 14,000 rpm at ambient temperature. The upper plasma layer was diluted with three parts of an acidic buffer solution (0.19 M acetic acid and 10.8 mM sodium acetate trihydrate; pH 3.5), resulting in a reaction mixture with a pH of ca 4. The mixture was incubated with potassium fluoride to a final concentration of 250 mM for 15 min at 25°C.

Solid-phase extraction of sarin from plasma samples. Sarin was isolated from the acidified plasma samples (vide supra) by means of solid-phase extraction. The SepPak C₁₈ cartridge (type Classic; Waters, Milford, MA) was preconditioned by rinsing once with 4 ml of ethyl acetate and twice with 5 ml of water. A 25- µl sample of an internal standard solution (O-[U-²H]isopropyl methylphosphonofluoridate, d7-sarin, in ethyl acetate) was added to the sample mixture. Next, the mixture was charged onto the cartridge. The cartridge was washed with 5 ml of water and dried with air. After adding 1.2 ml of ethyl acetate, sarin and the internal standard were eluted from the cartridge. The vial containing the eluate was placed in a dry ice/acetone bath in order to freeze out small droplets of water coeluting from the cartridge. The samples (ca. 600-800 µl) were stored in GC vials at -20°C until analysis.

Gas chromatographic analysis of sarin extracts from plasma samples. The analysis of sarin was performed by means of two-dimensional gas chromatography with large volume injection. Briefly, a Carlo Erba (Milano, Italy) 5300 Mega series gas chromatograph was equipped with a flame ionization detector (FID) and a nitrogen phosphorus detector (NPD), a MUSIC (Multiple Switching Intelligent Controller,' Chrompack/Varian, Middelburg, The Netherlands) and a Chrompack/Varian TCT (Thermal Cold Trap) injector. Helium gas is used as a carrier and is pressure regulated (110 kPa) for the analytical column (CP Wax 57, length 30 m; i.d. 0.25 mm; film thickness 0.5 µm). The precolumn (CP Sil 8 CB column; length, 30 m; i.d. 0.53 mm; film thickness 5 µm) is flow regulated (ca 16 ml/min). Flow rates of air and hydrogen through the FID and the NPD were 350 and 35 ml/min, respectively. Helium was used as make-up flow (38 ml/min) for the NPD. The temperature of the detector bases was set at 250°C, whereas the temperature of the injector base was set at 200°C.

The sample was charged in portions of 100 µl onto the preconditioned TCT sample tube (length 10 cm; i.d. 3 mm) filled with ca. 100 mg of Tenax TA (60-80 mesh) and blown dry with a flow (250 ml/min) of nitrogen. For large samples, up to 500 µl, the tube was purged for 45 min. For desorption the TCT oven is heated for 6 min at 180°C, while the TCT trap (a medium polar deactivated retention gap, i.d. 0.53 mm) is cooled with liquid nitrogen (-90°C). The compounds of interest that were desorbed from the Tenax TA are collected on this trap and injected onto the precolumn by flash heating the trap (from -90 to 200°C at a rate of ca 10°C/s, 12 min constant at 200°C). During precolumn analysis the oven temperature is kept contant at 70°C for 6 min and programmed to 90°C at a rate of 10°C/min. The MUSIC controller and valve box are set for trapping sarin and internal standard by means of "Deans switching." The fraction is collected at -70°C in the MUSIC trap and reinjected at 200°C on the analytical column after cooling down the GC oven to 70°C. The temperature of the oven is programmed from 70 to 220°C at a rate of 5°C/min and kept constant for 15 min. The peaks of internal standard and sarin are resolved completely on the analytical column and detected with the NP detector. The absolute detection limit (S/N = 2) for sarin was approximately 0.15 pg depending on the noise (500 µl injection of 0.3 pg/ml sarin in ethyl acetate). All columns were purchased from Chrompack/ Varian. Data acquisition was performed with the data acquisition software program Harley Systems Peak Master ('s Hertogenbosch, The Netherlands), which runs on a PC.

Calibration of the system was performed on a daily basis. The response factor was stable during the various experiments. Over a period of several months the variation coefficient was less than 10%. The calibration curve was linear in the concentration range used for the analyses (correlation coefficient > 0.999), also with a relatively large amount of internal standard (47 pg) added to the samples. In order to check the sample work-up, guinea pig blood (EDTA) was spiked with sarin (188 pg/ml) and worked up as described before. The analyzed concentration was 67% of the spiked level using the internal standard method.

Before analyzing sample extracts, a blank solvent was injected and checked for interfering peaks. In order to exclude any memory effects of the Tenax tubes, these were washed with a few milliliters of ethyl acetate and heated under a stream of helium (>_14h, 220°C). After this cleaning step, the tubes were capped. The extracts were stored at -20°C, separated from standards. Also the internal standard solution was stored separately to prevent contamination.

Administration of pyridostigmine bromide. In order to determine the LOEL for vehicle and pyridostigmine- pre-treated guinea pigs and marmosets, Alzet osmotic minipumps (Model 2002; Alza Corp., Palo Alto, CA) containing either vehicle (20% propylene glycol, 10% ethanol, 70% water containing 1 part glacial acetic acid in 2000 parts distilled water) or pyridostigmine bromide dissolved in vehicle were implanted subcutaneously under halothane N₂O anesthesia 4 days before the sarin exposure started. From pilot experiments it is known that released doses of pyridostigmine of 0.04 and 0.02 mg/kg/h induce approximately 30% inhibition of the ChE activity in blood of guinea pigs and marmosets, respectively. This pretreatment with pyridostigmine was continued during the exposure to sarin vapor.

Assessment of the LOEL for sarin exposure. A cannula was installed in the left carotid artery of the guinea pig. After recuperation the animal was restrained in order to take blood samples from the cannula (and to take photographs of the eyes for measurement of miosis). For this purpose, a metal tube-like grid to enclose the animal was mounted on a floor grid. Additionally, a metal neck-bow as well as a "jaw-bone print" constructed from synthetic material were necessary to optimize restrainment of the animal (Van Helden et al., 2001). In order to determine the LOEL for guinea pigs (n = 5; Gpl-Gp5) one conscious animal per day was exposed to sarin at a concentration of 0.05-1.0 µg/m³ during a period of 5 h. During exposure blood samples (400-800 µl) were taken from the carotid cannula every 30 min for measurement of the internal dose. The same volume of saline was given back via this cannula.

Experiments with four marmosets (Ml-M4) were similar to those with guinea pigs, except that (1) the conscious marmosets were restrained during exposure to sarin in a metal chair, with arms and legs fixed with tape on the chair and wearing a plastic helmet in order to fix the head to the chair (Van Helden et al., 2001) and (2) the volume of blood samples taken for internal dose measurements was smaller than in the case of guinea pigs, i.e., 200-500 µl. The animals had learned to sit in the exposure chamber for several hours while watching video films featuring marmosets. Film watching appeared to be necessary to keep the marmosets awake.

Fig. 1. Schematic diagram of equipment for sarin vapor generation in air (right), exposure (left), and analysis (upper small panel). See text for detailed description. The sarin vapor is generated, diluted, and humidified before transport through a heated interface and routed to the animal exposure chamber. Vapor samples for analysis are taken just before the animal exposure chamber. The samples are then transported to the Cold Sample Trap (CST) for sample enrichment after which the sample is directly injected into the GC for analysis. I–IV are three-way valves.

Long-term low-level exposure studies to sarin at concentrations 0.05 µg/m³ require sensitive techniques for analysis and control of the vapor concentrations. Therefore a novel configuration was developed, involving generation of the vapor, the construction of the exposure chamber, and the appropriate analysis system. Fig. 1 gives a schematic view of the system.

In order to obtain the desired concentration of sarin vapor in air, cooling of the rather volatile agent in the evaporation vessel down to ca. 5°C was needed, as well as two dilution steps (dilution factor 50-100). Attention had to be paid to prevent pressure build-up, cold spots, and the occurrence of chemically active sites since these factors contribute to undesired fluctuations of the vapor concentration. Where possible glass tubing was used, which was thermostatted either directly by using thermostatted tubing or by packing with thermostatted material. Two types of overpressure safeties were used (see Fig. 1), mainly as safety precautions in combination with manometers in the nitrogen and air supply and on the exposure chamber (type 1) but also to ensure a constant pressure gradient in the system (type 2), thus allowing a constant vaporization and dilution of the agent.

The linearity in the change of the vapor concentration with the "turns" of the needle valve that regulates the degree of dilution in the mixing chamber is influenced by active sites and diffusion effects in a rather unpredictable way, i.e., a sudden concentration drop or a long-lasting high concentration can occur. Also memory effects due to diffusion are rather pronounced, especially if the flow through the needle valve is rather low. However, by using two dilution steps, the overall "agent-flow" through the system can be in-creased, thus minimizing the memory or diffusion effects.

The guinea pig or marmoset is placed inside the exposure chamber, while the animal is provided with a carotid cannula for taking blood samples. By opening the valves I to IV, the animals are exposed to sarin vapor, at a total vapor flow through the chamber of ca 5 L/min. The vapor is vented by using a vacuum pump after being led through a carbon filter. During the exposure the vapor is sampled just in between valve II and the exposure chamber and directed to the GC for analysis. The pressure in the chamber is kept at 0.5 kPa (5 mBar). During handling of the animals the vapor is by-passed by switching the valves I to IV and the chamber is flushed with clean air.

Several techniques are available for vapor sampling and analysis. Among these, a fixed volume gas sampling valve in combination with gas chromatographic analysis is in most cases satisfactory. However, the minimum detectable concentration with such a system is ca. 0.1-1.0 mg sarin/m³ when using an alkali flame (NP) detector in combination with the usual volumes (<1 ml) of the sampling loop. In order to collect ca 1 pg of sarin, which is close to the absolute detection limit of the NP detector, from the highly diluted exposure air, unrealistically large injection volumes should be used. Off-line sampling methods, e.g., with a solid or liquid adsorbent, allows a preconcentration of a larger sample volume. However, on-line detection is preferred because the process can be monitored more adequately.

A new analytical configuration was constructed in which vapor samples were concentrated in a cold trap during 0.8 min, followed by flash heating and analysis by means of gas chromatography with NP detection. In this way sarin levels > 0.05 µg/m³ could be analyzed semicontinuously at 4-min time intervals. The gas chromatograph was equipped with an on-column injector and an externally controlled sampling device. This device was constructed in house by using a Valco (Schenkon, Switzerland) six-port injection valve with an electronic actuator, the cold trap and Music communicator of the MUSIC system and in-house constructed hard-ware boxes for data communication. A combination of uncoated deactivated fused silica tubing and a very short analytical column was used to perform gas analysis of sarin and calibration of the GC on the same detector. A DB 1 column (length 10 m; i.d. 0.32 mm, film thickness 1 pm) was installed onto the on-column injector and connected to an all glass Y- press fit connector (see Fig. 1, gray spot in the GC). The gas sampling valve was connected to the other "leg" of this Y-press fit with a piece of uncoated deactivated fused silica (length 40 cm; i.d. 0.32 mm). Finally, 20 cm of a DB 1 column (i.d. 0.32 mm; film thickness 1 µm) was used to connect the Y-press fit to the NP detector.

Just before the entrance of the exposure chamber, a vapor sample is taken from the main stream by means of a piece of PEEK tubing and directed through a six-port Valco sampling valve. Connected to this valve, a piece of uncoated and deactivated fused silica (i.d. 0.32 mm; length 60 cm) is led through the metal cold trap of the MUSIC apparatus and guided back (Cold Sample Trap, CST, see Fig. 1). The flow in the cold trap (-70°C) is kept constant at 20 ml/min during the sampling period by means of a needle valve and pump.

Fig. 2. Chromatograms of three consecutive analyses of sarin at 4-min intervals from a vapor stream containing sarin at a level of ca 0.1 µg/m3 (-16 ppt) via the cold trap technique. The sampling time in the cold trap was 0.8 min at a vapor stream of 20 ml/min. See text for further details.

After the trapping period the valve is switched and the trap is heated rapidly to 120°C. The trapped components are injected into the connected column and finally detected with the NP detector. Fig. 2 shows a typical chromatogram of the vapor analysis. The gas chromatograph was calibrated by on-column injection of 1 µl of standard solution containing sarin in ethyl acetate in concentrations varying from 0.1 to 10 ng/ml. In order to validate the cold trapping technique for low levels of sarin, a different and independent sampling technique was used involving adsorption on solid Tenax material. For the latter approach, the PEEK tubing that led to the sampling valve was connected to a sampling tube filled with Tenax TA. Next, during a fixed period of time and using a fixed sample flow, the vapor is trapped on this Tenax. Subsequently, the tube is capped and transferred to a Carlo Erba Gas Chromatograph equipped with a TCT injector, a two- dimensional column switching system, MUSIC, and NP detection, as used for sarin analysis in biosamples (see Materials and methods).

For validation purposes, a sarin vapor concentration (ca. 0.1 µg/m³) was generated and analyzed alternately with the CST and the TCT procedure. See Fig. 3 for a schematic representation of the procedure. Numbers between brackets refer to sampling numbers in Fig. 3. After_, five analyses by means of the CST technique [1] the sample tube was disconnected from the GC valve and connected to the first [2] of two Tenax sample tubes. The sample was passed through the Tenax for 10 min at a flow rate of 100 ml/min. Next, the tubing was disconnected and connected to the GC valve for another set of CST analyses [3]. Once again the tubing was disconnected from the gas valve and connected to the second Tenax sample tube [4] and the vapor was charged onto this tube. Finally, the tubing was disconnected from the tube and connected to the gas valve for the last set of CST analyses [5]. The concentration of sarin in the vapor deter-mined with the CST was calculated by averaging the individual vapor concentrations in each of the time slots 1, 3, and 5. Results of the measurements as schematized in Fig. 3 are given in Table 1. The difference between the on-line cold- trapping sampling technique and the off-line analysis with the TCT-MUSIC configuration is approximately 10% at these levels of ca. 0.1 µg/m³ sarin in air.

Fig. 3 Validation of the `cold sample trapping' (CST) approach for analysis of sarin vapor. Scheme of the sampling periods used for analysis of the concentration of sarin vapor determined with the CST technique [1] before the first Tenax/TCT sampling, [2] the 10-min sampling period for the first Tenax/TCT analysis, [3] in between the two Tenax/TCT samplings, [4] the 10-min sampling period for second Tenax/TCT analysis, and [5] after the second Tenax/TCT sampling period. Time points at which exposure concentrations were actually measured over an 0.8-min period of time are indicated (•).

Fig 4. Time course of generated vapor concentrations of sarin in air during a 300-min exposure of Gp3. The vertical arrow indicates the first time point at which the internal dose of fluoride-regenerated sarin could be analyzed by means of two-dimensional gas chromatography (S/N 2). Time points at which exposure concentrations were actually measured over an 0.8 min period of time are indicated (•).

One animal per day was whole-body exposed to sarin vapor for a period of 5 h. During the exposure the concentration of sarin vapor in the exposure air was measured on line by means of the CST technique every 4 min. In this way, the mean exposure dose (Ct) can be calculated semicontinuously during the exposure period. Fig. 4 gives a representative example of the measured exposure concentrations in the course of the 300-min exposure period for Gp3. Some apparent large fluctuations in the exposure concentration are due to occasional misintegration of the sarin peaks during the high-speed repetitive injections. Blood samples (400-800 µl) were taken every 30 min for internal dose assessment by analysis of sarin regenerated with fluoride ions from covalent binding sites in plasma. For the latter measurements, gas chromatographic data were only accepted if the signal to noise ratio (S/N) was >_2. The measured exposure doses and internal doses of sarin in Gps 1-5 are collected in Table 2.

Table 1 Validation of the on-line gas chromatographic analysis of sarin vapor in air with the cold trap sampling technique by means of off-line adsorption on Tenax and subsequent thermal cold trap analysis

Sampling	 Concentration of sarin (µg/m3)   
Period		CST analysis	Tenax/TCT analysis
1 		0.15±0.01 
2				0.09
3 		0.08±0.01 
4				0.13
5 		0.08±0.01
Mean 		0.10±0.05	0.11±0.02

Note. Values are means ± SEM, n 5. CTS, cold trap sampling; TCT, thermal cold trap.

The shaded data in Table 2 pertain to the internal doses from which LOEL values are derived, i.e., measured at the earliest time points at which the internal doses in the course of the 300- min exposure could be measured with S/N 2, and satisfied the latter criterion at all subsequent time points at which internal doses were measured. Exposure periods needed to reach these LOEL values varied between 30 and 120 min. Time- averaged concentrations of sarin vapor were used to calculate the mean concentration over a particular time period of exposure. The individual LOEL doses of exposure were calculated on the basis of these values, as summarized in Table 3.

The average LOEL for Gps 1-5 was calculated to be 0.010 ± 0.002 mg.min/m³ (mean ± SEM; n = 5). A similar series of exposures as with Gps 1-5 was performed with five Gps that were continuously infused with pyridostigmine bromide by means of an implanted Alzet pump, resulting in ca. 30% inhibition of erythrocyte AChE. The average LOEL in these animals was 0.014 ± 0.003 mg.min/m³_. This value is not significantly different (p < 0.05) from that in vehicle-pretreated guinea pigs (data not given; see Van Helden et al., 2001).

Table 2 Mean exposure concentrations and corresponding internal doses for whole body exposure of male guinea pigs (Gp's 1–5) to sarin vapor in air over a 5-h period

	Gpl			Gp2			Gp3			Gp4			Gp5	
EP	MC	    IP		MC	      IP	MC		IP	MC		IP	MC		IP
0	0		--a	0		--a	0		--a	0		--a	0		--a
0-30	0.11± 0.02	--a	0.43± 0.07    43	0.12±0.02	--a	0.04±0.01	--a	    --a		--a
0-60	0.11± 0.02	--a	0.39± 0.04    66	0,14±0.02    9.8	0.04±0.01	--a	0.02±0.01	--a
0-90	0.12±0.01	--a	0.36± 0.03	--b	0.14±0.01    12		0.05±0.01	--a	0.04±0.01    7.3
0-120 	0.14±0.01   20		0.36± 0.03	--b	0.14±0.01    16		0.06±0.01    4.7	0.05±0.01    13c
0-150	0.16±0.01    21		0.36± 0.02	--b	0.13±0.01	--b	0.07±0.01    5.5	0.06±0.01    10
0-180	0.16±0.01    32		0.38± 0.02    146	0.13±0.01	--b	0.09±0.01    9.2	0.06±0.01	--b
0-210	0.16±0.01    27		0.38± 0.02	--b	0.13±0.01	--b	0.10±0.01	--b	0.07±0.01	--b
0-240	0.16±0.01    34		0.38± 0.02	--b	0.13±0.01	--b	0.11±0.01    11		0.07±0.01	--b
0-270	0.16±0.01    35		0.39± 0.02	--b	0.13±0.01	--b	0.12±0.01	--b	0.08±0.01    16
0-300	0.15±0.01    30		0.41± 0.02    205	0.13±0.01    26		0.13±0.01    16		0.08±0.01	--b

EP = Exposure Period (min)
MC = Mean concentration (µg/m3)
ID = Internal dose (pg/ml plasma)

Note. Values for exposure concentrations are means ± SEM. Exposure doses were measured at 4-min intervals by 
      means of gas chromatography. Internal doses are expressed as amount of sarin regenerated by fluoride 
      ions from binding sites in blood samples, which were taken at 30-min intervals (pg sarin/ml plasma). 
      See text for further details.
a Below detection limit (S/N < 2).
b Not analyzed.
S/N = 1.9.

Experiments with marmosets M1-M4 were performed in a similar way as those with guinea pigs. Results of these measurements are collected in Table 4. Shaded data in Table 4 pertain to the internal doses from which LOEL values are derived, using the same criteria as for guinea pigs (vide supra). Exposure periods needed to determine the LOEL dose varied from 60-180 min.

The mean individual exposure concentrations of sarin (µg/m³) generated over the period of time between the start of the exposure and the time point at which the LOEL value could be established are given in Table 5. The individual LOEL levels of exposure were calculated on the basis of these values and ranged between 0.022 and 0.066 mg mini m³. The averaged LOEL level for marmosets (M1-M4) was calculated to be 0.04 ± 0.01 mg min/m³.

A similar series of exposures as with M1-M4 was per-formed with five marmosets that were infused with pyridostigmine bromide (vide supra), resulting in ca. 30% inhibition of erythrocyte AChE. The average LOEL in these animals was 0.050 ± 0.002 mg min/m³. This value is not significantly different (p < 0.05) from that in vehicle-pretreated marmosets (data not given; see Van Helden et al., 2001).

The surprisingly low exposure levels at which internal doses of sarin become measurable by means of fluoride-induced reconstitution of sarin from its binding sites in plasma, in combination with our desire to investigate low-level exposure in a realistic scenario, i.e., over a period of several hours, forced us to develop an apparatus in which exposures of small animals to sarin at the lower ppt level can be performed. Moreover blood samples had to be taken from the animals during exposure. Since it was not known at which point of time in the course of the 5-h exposure period an internal dose becomes measurable, it was mandatory to develop a system that allows the measurement of exposure concentrations as continuously as possible. How-ever, the requirement that concentrations of sarin in air >0.05 µg/m³, i.e., >0.05 pg/ml, should be quantified led to a compromise in which sarin in a controlled sample stream of air (20 ml/min) was collected in a cold trap during a period of 0.8 min, after which analysis was performed upon flash heating for gas chromatographic analysis with alkali flame (NP) detection. In this way exposure concentrations of sarin averaged over a 0.8- min period of time could be measured at 2- to 5-min intervals. This specific configuration permitted validation since the sarin vapor could also be conveniently adsorbed on a solid adsorbent (Tenax) followed by thermal desorption and an independent analysis by means of our earlier developed TCT/MUSIC two-dimensional gas chromatographic system (Benschop et al., 1987). Based on the data of the validation, it is concluded that the on-line method of cold trapping and flash heating (CST) is a reliable approach for routine measurements of vapor concentrations of sarin in air 0.05 µg/m³. In theory an almost unlimited volume of vapor can be concentrated into the cold trap for subsequent analyses at even lower levels. In practice, due to accumulation of contamination and humidity of the vapor, the maximum sample size is limited to 15–20 ml, which is sufficient for the purpose of the present investigations.

Table 3 Mean exposure concentration of sarin vapor in air generated over the period of time (time to LOEL) between the start of the exposure and the time point at which fluoride-regenerated sarin (internal dose) could be measured in guinea pigs by means of gas chromatography (S/N 2) and calculation of the individual LOEL

	   Guinea pig           Gpl      	Gp2      	Gp3      	Gp4      	Gp5      . 
	   Time to LOEL (min)	120		30		60		120		90	
Mean concentration to 		0.14±0.01	0.43±0.07	0.14±0.02	0.06±0.01	0.04±0.01
  time to LOEL (mg/m3/103)
LOEL (mg/min/m3)			0.017±0.001	0.013±0.002	0.008±0.001	0.007±0.001	0.004±0.001
Mean LOEL (mg/min/m3)						0.010±0.002

Note. Values are means ± SEM. Numbers of guinea pigs (Gps 1-5) correspond to those in Table 2.

Table 4. Mean exposure concentrations and corresponding internal doses for whole body exposure of marmosets (Ml-M4) to sarin vapor in air over a 5-h period

The measured LOEL values, based on the first reliable measurement of an internal dose in blood samples, appears to be approximately fourfold higher in marmosets than in guinea pigs. However, this difference reflects for a large part a technical problem emerging from drawing larger blood sample volumes from guinea pigs (500–700 µl) than from marmosets (200–500 µl), since overall more blood can be drawn from guinea pigs than from marmosets. Consequently, fluoride-regenerated sarin could be analyzed earlier in guinea pigs than in marmosets. If this difference is taken into account, the LOEL in marmosets is at most twofold higher than in guinea pigs. The set-up of our experiments did not allow determination of the LOEL values at the end of the 5-h exposure period. Instead, reliable LOEL values were measured after exposure periods of 30 to 120 min in guinea pigs and 60 to 180 min in marmosets. However, this range of time periods to LOEL suggests that LOEL measurements after a >5-h exposure period will be feasible.

Table 5 Mean exposure concentration of sarin vapor in air generated over the period of time (time to LOEL) between the start of the exposure and the time point at which fluoride- regenerated sarin (internal dose) could be measured in marmosets by means of gas chromatography (S/N 2), and calculation of the individual LOEL

					Marmoset  M1            M2		M3		M4
                              Time to LOEL (min)  60            180             90		60
Mean concentration to time to LOEL (mg/m3/103) 	  0.91±0.16	0.37±0.03	0.40±0.11	0.37±0.05
LOEL (mg/min/m3)                                  0.05±0.01	0.067±0.005	0.04±0.01	0.022±0.00
Mean LOEL (mg/min/m3)                                                      0.04±0.01

Note. Values are means ± SEM. Numbers of marmosets (M1-M4) correspond to those in Table 4.

It is elucidating to compare the present LOEL measurements with those based on classical measurements of residual cholinesterase activity. The LOEL value in marmosets (0.04 mg min/m3) corresponds with an average regeneration of sarin from binding sites in plasma of (6.2 ± 1.4) pg/ml (± SEM; n = 4; cf Table 4). Assuming that marmosets have approximately the same amount of BuChE in plasma as humans (80 pmol/ml; see Polhuijs et al., 1997), this value corresponds with ca. 0.05% inhibition of BuChE in the LOEL-exposed marmosets. This value should be compared with measurements of residual cholinesterase activity, which, due to individual variations and other uncertainties, indicate inhibition of the enzyme upon at least 20% depression relative to "normalized" values in humans (Polhuijs et al., 1997). Therefore, measurement of fluoride-regenerated sarin in plasma is two to three orders of magnitude more sensitive than measurement of residual cholinesterase activity. This outcome should be considered as a minimum value since the LOEL value in marmosets is a conservative value (vide supra). Moreover, a more rigid interpretation of the data is hampered by the lack of information on binding sites in plasma for sarin and other nerve agents other than BuChE. For example, it appears that albumin in human plasma also binds the nerve agents sarin and soman (Black et al., 1999), whereas it is well known that the substantial levels of carboxylesterases present in guinea pig plasma bind nerve agent as well (Maxwell et al., 1987). Acetylcholinesterase in humans is inhibited to approximately the same percentage as BuChE in the plasma upon respiratory expo-sure of humans to sarin (Grob and Harvey, 1958). However, the absolute amount of AChE in human blood is 27-fold less than that of BuChE, which renders AChE a less suitable target for measurement of LOEL values, unless preferential inhibition of AChE would occur, e.g., in the case of nerve agents other than sarin (Polhuijs et al., 1997). Recently, a method was developed in our laboratory in which both inhibited and uninhibited BuChE is isolated from plasma by means of affinity chromatography, followed by digestion of the enzyme with pepsin and mass spectrometric analysis of the peptide containing the active site serine (Fidder et al., 2002). This approach will exclude ambiguities as to which degree of fluoride regeneration of sarin from plasma may result from phosphylated sources other than BuChE. Based on the assumption that the observed regeneration of sarin stems largely from binding to BuChE, it is not surprising that pretreatment of the guinea pigs and marmosets with pyridostigmine, leading up to 30% inhibition of AChE, has no measurable effect on the observed LOEL since binding of this carbamate to BuChE will be at most ca 10% under these conditions (Thomsen et al., 1991).

The data that were collected in order to establish a LOEL value for sarin exposure are also valuable in investigations of the relationship between exposure dose and internal dose in a more general way. Analysis of the data in Tables 2 and 4 shows that, at least in the extremely low-dose range of our investigations, a reasonably linear relationship is observed between exposure dose and internal dose, albeit that substantial variations in the slope of the line are observed between individual animals. Representative illustrations are given in Fig. 5A and B for guinea pigs Gpl and Gp4 and for marmosets M1 and M4, respectively.

Fig. 5. (A) Correlation between the exposure dose (mg min/m3) and the corresponding internal dose (pg sarin regenerated with fluoride ions/ml plasma) for guinea pigs Gpl (o) and Gp4 (•); Gpl: Y = 491X + 12.4 (r² =-0.66); Gp4: Y = 332X + 2.58 (r² = 0.96), in which Y = pg regenerated sarin/ml plasma and X = exposure dose (mg min/m³). (B) Correlation between the exposure dose (mg min/m3) and the corresponding internal dose (pg sarin regenerated with fluoride ions/ml plasma) for marmosets Ml (o) and M4 (•); Ml: Y = 122X -0.9 (r² = 0.87); M4: Y = 176X -2.8 (r² = 0.91), in which Y = pg regenerated sarin/ml plasma and X = exposure dose (mg min/m3).

It is interesting to compare the LOEL values that we measured with recently published recommendations for occupational exposure limits for sarin vapor in air (Mioduszewski et al., 1998). A so-called "worker population limit" (WPL) was defined for workers without respiratory protection, i.e., a minimum averaged air concentration of 0.1 µg/m³, averaged over an 8-h working day. This would correspond with a no-effect Ct value of 0.048 mg min/m³. Moreover, a so-called short-term exposure limit (STEL) was defined corresponding with four exposures to sarin at a level of 2 µg/m³ for 15-min periods, which would lead to a Ct value of 0.12 mg min/m³ for the four STEL periods on 1 day. At these occupational limits even the mildest miosis or inhibition of AChE in blood should not occur. Our data on LOEL values suggest that these WPL and STEL limits should be reconsidered if indeed the slightest inhibition of cholinesterase should be prevented.

This work was supported by US Army Medical Research Acquisition Activity, Contract Number DAMD17- 97-1-7360.

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