Acrylaldehyde

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication report which meets basic scientific principles

Data source

Reference

Reference Type:

publication

Title:

Nasal Uptake of Inhaled Acrolein in Rats

Author:

Struve MF et al.

Year:

2008

Bibliographic source:

Inhalation Toxicology 20: 217-225

Materials and methods

Objective of study:

absorption

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The uptake efficiency of acrolein was measured in the isolated upper respiratory tract of anesthetized naive rats under constant velocity unidirectional inspiratory flow rates. An additional group of animals was exposed to acrolein for 14 days prior to performing nasal uptake studies.
Olfactory aud respiratory glutathione (GSH) concentrations were also evaluated in naive and acrolein-preexposed rats.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acrylaldehyde

EC Number:

203-453-4

EC Name:

Acrylaldehyde

Cas Number:

107-02-8

Molecular formula:

C3H4O

IUPAC Name:

acrylaldehyde

Details on test material:

- Name of test material (as cited in study report): acrolein purchased from Aldrich Chemical Company (Milwaukee, Wl, USA)
- Analytical purity: > 99%
- Impurities (identity and concentrations): 200 ppm hydroquinone
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratory (Kingston, NY, USA)
- Age at study initiation: 6 weeks
- Weight at study initiation: 190 +/-2.1 g
- Fasting period before study: no
- Housing: on direct bedding (Alpha-DriTM, Shepard Specialty Papers, Kalarnazoo, MI, USA) in filter-capped cages
- Diet: ad libitum, certified NIH-07 rodent chow (Zeigler Brothers, Gardners, PA, USA)
- Water: ad libitum, reverse osmosis water (HydroService and Supplies, Research Triangle Park, NC, USA)
- Acclimation period: two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18- 26
- Humidity (%): 30-70
- Air changes (per hr): no data on air changes, animal rooms were ventilated with HEPA-filtered air
- Photoperiod (hrs dark / hrs light): 12-hr light-dark cycle

Administration / exposure

Route of administration:

inhalation: gas

Vehicle:

other: nitrogen

Duration and frequency of treatment / exposure:

80 minutes

Doses / concentrations

Remarks:

Doses / Concentrations:
- test on naive rats: 0.6, 1.8, or 3.6 ppm under constant-velocity unidirectional inspiratory flow rates of 100 or 300 ml/min
- test on preexposed rats
-- preexposure: 0.6 or 1.8 ppm, 6 h/day, 5 days/wk, for 14 days prior to performing nasal uptake studies
-- nasal uptake study: 1.8 or 3.6 ppm at a 100 ml/min airflow rate

No. of animals per sex per dose / concentration:

6

Control animals:

yes, sham-exposed

Details on study design:

Animals were anesthetized with urethane (Acros Organics, MorrisPlains, NJ) at approximately 1.3 g/kg (ip). Anesthetized rats were placed on a heated pad (SnuggleSafe, South Holland, IL) in a supine position, and the trachea was surgically exposed by blunt dissection using methods described by Morris (1999). A 2.5-cm polyethylene endotracheal tube (PE 205, Clay Adams; Parsippany, NJ) was positioned toward the lungs while a 14-G
catheter (4058-20 Jelco, Cincinnati, OH) was inserted into a second tracheal incision so that the catheter tip was positioned near the larynx. The animal's head and nose were placed in an anesthesia nose-only cone (Euthanex Corp., Palmer, PA) that was used to deliver acrolein. Nasal uptake was measured under constant velocity unidirectional inspiratory flow at rates approximating 67 and 200% of the predlcted minute ventilation (150 ml/min) of the adult male rat. Air was drawn through the isolated URT for up to 80 min under the desired flow conditions. Anesthesia was monitored and administered as needed throughout the exposure period. Animals were killed via exsanguination immediately after the end of exposure and tissues collected within 5-10 min.

Glutathione Measurement
Immediately following the end of the acroleine exposure, anesthetized rats were killed by decapitation, and the head was sectioned sagitally on the bridge of the nose. The nasal respiratory and olfactory mucosa with the supporting turbinates were visually identified and dissected from the nasal cavity. Freshly harvested tissue (40-100 mg of mucosa and turbinate) was placed directly into 400 pµl cold 5% meta-phosphoric acid (MPA) and homogenized. Separate 100-µl aliquots of the homogenate were used for measurement of total protein, total glutathione, and GSSG. The GSSG aliquot also inincluded 5 µl of the glutathione scavenger 1-methyl-2-vinylpyridinium trifluoromethanesulfonate (M2VP). All aliquots were frozen at - 80°C until analysis.

Data analysis
The concentration of acrolein entering the URT (pre-nose) and exiting the URT (post-nose) was measured approximately every 13 min after initiation of flow through the URT through the end of the 80-min acrolein exposure. More frequent sampling (e.g., every 3-6 min) were performed during shorter uptake experiments. Inhalation uptake efficiency (UE) was calculated as (pre-nose sampie - post-nose sample)/pre-nose sample) x 100, and expressed as a percentage. The grand mean UE was calculated as the overall average of the six samples for each animal.

Details on dosing and sampling:

Acrolein Vapor Generation and Characterization:
Test atmospheres of acrolein were generated with certiefed gas cylinders containing either 40 or 80 ppm acrolein in nitrogen. Acrolein exposure concentrations were generated by metering acrolein in nitrogen from the gas cylinder through a mass flow controller (MKS Instruments, Andover, MA) into a "T" in the dilution airflow, where the acrolein was mixed with dilution air to achieve the target concentrations. The dilution air was drawn from an instrument grade compressed air supply, a portion of which was humidified. The acrolein exposure concentration was controlled by changing the acrolein delivery rate and the dry and humidified airflow rates to achieve the desired concentration. Total flow rates through the system were maintained at 300 to 800 mI/min for URT flow rates of 100 or 300 ml/min, respectively. Acrolein-laden, humidified air (to 45-55% relative humidity) was drawn continuously through the isolated URT with a rotary-vane vacuum pump (GAST Manufacturing, Benton Rarbor, MI). The test atmosphere was presented to the breathing zone of the animal at an excess flow rate at least 100 ml/min above the projected URT flow and sampling flow rate. The average air temperature and relative humidity maintained during the exposures ranged from 21.7 to 23.0°C and from 45 to 55%, respectively. The exposure portion of the system was contained within a vented portable hood.

Air sampIes were analyzed approximately every 3 min. The sampling system used to draw samples consisted of Teflon tubing and fittings. Two tees were used to connect the sampling system to either the test atmosphere near the breathing area in the rat nose cone (pre-nose) or the endotracheal tube (post-nose). SampIes were drawn through a multiport gas sampling valve connected to a gas chromatograph (GC) at a flow rate of 30 ml/min from either location. Sampling airflow rates were controlled with rotameters and needle valves, which were verified using a BIOS DC-I flow calibrator (Bios International Corporation, Pompton Plains, NJ). Acrolein concentrations were measured with a Hewlett Packard 5890 Series II GC (Agilent Technologies, Palo Alto, CA) equipped with a flame ionization detector (FID) and an oven temperature of 90-100 °C. For the 1.8-ppm and 3.6-ppm exposures, a 5% phenyl-/95% methylpolysiloxane column (EC5, Alltech Chromatography, Deerfield, IL) was used (elution time approximately 0.64 min), while a packed column (10% FFAP on CWAW 80/100, Alltech Chromatography, Deerfield, IL) was used for the 0.6-ppm acrolein exposures (elution time approximately 1.36 min). An eight-position gas sampling valve was used by the GC to sample at a programed sequence of locations from both pre and
post-nose locations from both exposure systems. The GC was calibrated with standards made by injecting certified high-purity acrolein (3000 ppm) into Tedlar gas sampling bags (SKC, Inc., Eighty-Four, PA), which were further filled with a measured volume of instrument-grade air to achieve the desired range of concentrations. Area counts were converted to concentrations in parts per million of acrolein.

Statistics:

To test for the effects of acrolein concentration, airflow rate, and exposure time, and also their interactions, experimental data were analyzed by a three·factor multivariate analysis of variance for a design with repeated measures. If the concentration or flowrate effect was significant, and the effect was not involved in a significant interaction, Tukey's multiple comparison procedure for the overall mean UE data was used to determine which concentrations and/or flow rates were different. Statistical analyses were performed with JMP Statistical Software (SAS Institute, Inc., Cary, NC). A probability value of .05 was used as the level of significance for all statistical tests. Unless otherwise indicated, all data presented represents mean ± standard error ofthe mean (SEM).

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Nasal Uptake of Acrolein in Naive Animals:
URT uptake efficiency of acrolein was dependent on the concentration of inspired acrolein, duration of exposure, and airflow rate. Overall average
UE was significantly greater at the lowest concentration than at either of the higher concentrations (0.6 > 1.8, 3.6 ppm) (p < .0001, Tukey's test). At a 100 ml/min airflow rate, mean UE of inspired concentrations of 0.6 ppm acrolein (97.8 ± 1.5%) was nearly double the efficiency of uptake of 3.6 ppm (49.5 ± 4.8%). A general decline in UE during the 80min exposure was observed and a repeated-measures analysis of variance (ANOVA) revealed time as a significant effect on uptake effieiency (p < .001). For example, animals exposed to an inspired concentration of 3.6 ppm acrolein had an approximate 50% decrease in UE during the 80-min exposure. Significant interactions between acrolein concentration and airflow (p =.95) and time and flow rate (p =.12) were not observed. A significant interaction between acrolein concentration and time (p = .0l) was, however, observed.



Nasal Uptake of Acrolein in Acrolein Preexposed Animals:
Some animals were exposed to 0.6 or 1.8 ppm acrolein, 6 h/day, 5 days/wk, for 14 exposure days prior to performing acrolein nasal uptake studies at a single airflow rate (100 ml/min). As with naive animals, URT uptake efficiency of acrolein was time dependent (p < .0001). URT uptake efficiency of acrolein in pre-exposed animals was also dependent on the acrolein concentration used prior to (p = 0.01) and during the uptake exposure (p = .06). Animals that were pre-exposed to acrolein had higher UE than did naive animals exposed to air. Overall mean VE of acrolein in preexposed animals was approximately 13 to 38% higher than in air-exposed controls. A significant interaction between these two acrolein concentrations was not observed (p = .42).

Metabolite characterisation studies

Metabolites identified:

no

Details on metabolites:

The identification of metabolites was no object of the study, but Glutathion (GSH) levels following exposure were measured.

Nasal GSH Levels Following Short-Term Acrolein Inhalation by Naive Animals:
Nasal epithelial GSH concentrations were dependent on the concentration of inspired acrolein (p = .0002), airflowrate (p = .0007), and epithelial subtype (olfactory vs. respiratory; p = .0577). The duration of exposure did not have an overall significant effect on end-of-exposure epithelial GSH concentrations (p = .226). The duration of exposure had a marginally significant effect on end-of-exposure epithelial GSH concengtrations (p = .0705). Exposure to acrolein vapor also reduced GSH concentrations in the respiratory epithelium. In contrast, the study authors did not observe acrolein-induced GSH depletion in the olfactory epithelium of naive rats. The relative amounts of GSH and GSSG (expressed as the GSH/GSSG ratio) were dependent on the concentration of inspired acrolein (p = .0007), airflow rate (p = .053), and epithelial subtype (olfactory vs. respiratory; p < .0001).

Nasal GSH Levels Following Combined Short-Term Acrolein Inhalation and Acrolein Preexposure:
Nasal epithelial GSH concentration was dependent on the concentration of acrolein used during the preexposure (p = .006) and epithelial subtype (olfactory vs. respiratory; p < .0001). The acrolein concentration used for the uptake experiment did not have an overall significant effect on end-of- exposure epithelial GSH concentrations (p = .97). The relative amounts of GSH and GSSG (expressed as the GSH/GSSG ratio) were dependent on the epithelial subtype (olfactory vs. respiratory; p < .0001) but were only marginally dependent on either the concentration of inspired acrolein (p = .097) or the acrolein concentration used prior to the uptake experiment (p = .095).

Applicant's summary and conclusion

Executive summary:

The uptake efficiency (UE) of 0.6, 1.8, or 3.6 ppm acrolein was measured in the isolated upper respiratory tract (URT) of anesthetized naive rats under constant-velocity unidirectional inspiratory flow rates of 100 or 300 ml/min for up to 80 min. An additional group of animals was exposed to 0.6 or 1.8 ppm acrolein, 6 h/day, 5 days/wk, for 14 days prior to performing nasal uptake studies (with 1.8 or 3.6 ppm acrolein) at a 100 ml/min airflow rate. Olfactory and respiratory glutathione (GSH) concentrations were also evaluated in naive and acrolein-preexposed rats. Acrolein UE in naive animals was dependent on the concentration of inspired acrolein, airflow rate, and duration of exposure, with increased UE occurring with lower acrolein exposure concentrations. A statistically significant decline in UE occurred during the exposures.

The highest URT uptake efficiency of acrolein was seen in rats exposed to 0.6 ppm at a 100/min air flow rate and was 97.8 ± 1.5%.

Exposure to acrolein vapor resulted in reduced respiratory epithelial GSH concentrations. In acrolein-preexposed animals, URT acrolein UE was also dependent on the acrolein concentration used prior to the uptake exposure, with preexposed rats having higher UE than their naive counterparts. Despite having increased acrolein UE, GSH concentrations in the respiratory epithelium of acrolein preexposed rats were higher at the end of the 80 min acrolein uptake experiment than their in naive rat counterparts, suggesting that an adaptive response in GSH metabolism occurred following acrolein preexposure.