Registration Dossier

Administrative data

Endpoint:
specific investigations: other studies
Remarks:
Acute inhalation RD50
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
The experimental design is based on the procedures outlined in the Standard Test Method for Estimating Sensory Irritancy of Airborne Chemicals (ASTM designation: E 981-04) but differed in the type of exposure and plethysmography equipment used.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Principles of method if other than guideline:
This study is not designed to fulfill the requirements of any specific guideline. It is, however, based on the ASTM E 981-04: Standard Test Method for Estimating Sensory Irritancy of Airborne Chemicals (ASTM, 2004).
GLP compliance:
yes
Type of method:
in vivo
Endpoint addressed:
other: sensory irritation

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test Material Name: n-Propyl Propionate
Supplier, City, State (Lot, Reference Number): Chemical Marketing Concepts, New Milford, Connecticut (lot # WA3101FTB3)
Specific details on test material used for the study:
Test Material Name: n-Propyl Propionate
Chemical Name: Propyl ester propanoic acid
Synonyms: Propyl propionate, Propyl ester propionic acid
Supplier, City, State (Lot, Reference Number): Chemical Marketing Concepts, New Milford, Connecticut (lot # WA3101FTB3)
Purity/Characterization (Method of Analysis and Reference): Non-GLP characterization lists the purity of lot WA3101FTB3 as 99.96% with 0.02% water, and a specific gravity of 0.8825. The test material was not GLP-characterized.

Test animals

Species:
mouse
Strain:
Swiss Webster
Sex:
male
Details on test animals and environmental conditions:
Species and Sex: Mice (male)
Strain and Justification: Swiss Webster (CFW) mice. Selection of this strain and species was based on a variety of considerations including hardiness, low incidence of respiratory disease, and large preexisting database of RD50 determinations using this mouse strain. The Swiss Webster mouse is identified as the rodent of choice in ASTM Method E981-04. Although any mouse of the proper size could be used, marked differences in irritant sensitivity have been observed between different strains and sexes (ASTM, 2004).
Supplier and Location: Charles River Laboratories Inc. (Portage, Michigan)
Age at Study Start: Animals were approximately five weeks at arrival and approximately 6-8 weeks at the time of exposure.

Physical and Acclimation:
Each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International -AAALAC International). The animals were housed two-three per cage in stainless steel cages, in
rooms designed to maintain adequate environmental conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study. Mice were acclimated to the restrained whole body plethysmograph chambers for a half hour and one hour, two and one days prior to exposure, respectively.

Housing:
After assignment, animals were housed one per cage in stainless steel cages. Cages had wire mesh floors and were suspended above absorbent paper. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system.
Temperature: 22 ± 1°C
Humidity: 40-70%
Air Changes: 12-15 times/hour
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Randomization and Identification:
Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric
identification numbers.

Feed and Water:
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Feed and municipal water was provided ad libitum except during exposure and acclimation to the restrained whole-body plethysmography chambers. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained
from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained at Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Animal Welfare:
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities to be used for this study were Immuno 09 and Animal ID 01.


Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
air
Details on exposure:
Route, Method of Administration, Frequency, Duration and Justification:
Inhalation was selected as the route of administration since it is a potential route of human exposure.

Summary:
Separate groups of four mice were exposed to a range of n-propyl propionate vapor concentrations which produced responses (decreased respiration rate) ranging from a smaller effect (≤ 30% decrease) to the maximum (≤ 87% decrease) that could be obtained with n-propyl propionate.
For each exposure concentration a group of 4 male Swiss Webster mice were placed into restrained whole-body plethysmograph (WBP) chambers supplied with filtered room air. Mice were allowed a ten-minute acclimation period before the exposure/monitoring cycle began. This acclimation period was necessary to reduce spurious noise in the restrained WBP pressure signal due to random movements of the mouse. After the ten-minute acclimation period
the exposure/monitoring cycle was initiated and baseline respiratory rate data (breaths/minute) were collected for a ten-minute period. Test material exposure was then initiated and mice were exposed for 30 minutes with continuous collection of respiratory rate data. However, the 3063 ppm and 6793 ppm exposures were discontinued after 17 and 16 minutes, respectively, due to animal death. In order to assure that a maximum response (at that exposure concentration) was obtained, a plateau of response for at least 1 minute was obtained. The minimum decrease in respiratory rate considered significant was 12% (of baseline), provided that it was sustained for 3 minutes or reproducible in 3 animals for at least 1 minute (ASTM, 2004). In no case was the decrease in respiratory rate allowed to exceed 90% of the baseline value regardless of irritant concentration or potency (ASTM, 2004).
Artifacts in pressure changes due to movements of animals within the plethysomgraph chamber were electronically eliminated from the data set by the software system utilized.
A ten-minute recovery period was recorded after the end of exposure. At the end of the recovery period, recording was stopped and the animals were removed from the plethysmograph chambers and returned to their home cages with free access to feed and water. The mice were held for 7 days. Body weights and clinical observations were monitored to assess any untoward effects of exposure.

Exposure Chambers:
The restrained whole-body chambers are comprised of a reference and an animal chamber. Data are generated during normal respiration which creates a cyclic pressure increase/decrease in the animal chamber that is detected by a differential pressure transducer. Breathing frequency and volumes are measured using the timing and magnitude of the pressure changes in the animal chamber. Restrained whole-body plethysmography allows for continuous
data collection on conscious animals, and multiple endpoints can be evaluated. Based on the 4 liter per minute flow rate, the theoretical equilibrium time to 99% (T99) of the target concentration was 0.2 minutes.

Generation System:
ATedlar bag (SKC, Inc., Eighty Four, Pennsylvania) was filled with vaporized test material in air to serve as the stock gas bag used to generate the desired chamber concentration. Desired chamber concentrations were then generated using a sealed pump with a sampling line connected to the inlet valve of the stock gas bag. The pump delivered the test material at a set rate to the inlet of the mixing chamber, where the gas was mixed with the appropriate amount of dilution air.

Pretreatment Exposure Groups:
Additional exposures with and without capsaicin (stimulates trigeminal C-fiber sensory neurons) and theophylline (Adelta receptor antagonist) pretreatments were conducted that supported n-propyl propionate is a sensory irritant partially mediated by capsaicin-sensitive TRPV1 Cfibers. These data are excluded from the study interpretation as it is not relevant to the calculation of RD50.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure Concentration:
Test atmosphere concentration of the mixing chamber was continuously monitored with a Miran 1A infrared (IR) spectrophotometer (Foxboro/Wilks, South Norwalk, Connecticut). The IR was calibrated and a standard curve compiled, prior to the start of the exposure with standards of test material in Tedlar sampling bags (SKC, Eighty Four, Pennsylvania) to cover the range of exposure concentrations to be tested. The analytical concentration during each exposure was interpolated from the standard curve. The IR was checked prior to each exposure with a standard of known concentration.
The nominal concentration was calculated based on the amount of test material fed into the generation system and the total chamber airflow.
Duration of treatment / exposure:
30 minutes
Frequency of treatment:
Single 30 minute exposure
Post exposure period:
7 days
Doses / concentrationsopen allclose all
Dose / conc.:
246 ppm
Dose / conc.:
485 ppm
Dose / conc.:
542 ppm
Dose / conc.:
740 ppm
Dose / conc.:
1 214 ppm
Dose / conc.:
3 063 ppm
Dose / conc.:
6 793 ppm
No. of animals per sex per dose:
4 males/dose group
Control animals:
no

Examinations

Examinations:
Animal Observations and Criteria of Response:
A cage-side examination was conducted at least once a day, preferably at approximately the same time each day (usually in the morning). This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that would be observed include, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular
function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. Moribund animals not expected to survive until the next observation period were humanely euthanized that day. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

Clinical Examinations:
A hands-on evaluation of skin, fur, mucous membranes, respiration and nervous system function (including tremors, convulsions, diarrhea and animal behavior) was conducted during the preexposure period (prior to exposure on day 1), and on days 2, 4, and 7, with the exception of the 542 ppm exposure group in which these activities were inadvertently not done. On the day a clinical examination was conducted, the AM or PM cage-side examination may not have been conducted due to the more thorough nature of the clinical examination.

Body Weights:
All mice were weighed during the pre-exposure period (prior to exposure on day 1), and on days 2, 4, and 7, with the exception of the 542 ppm exposure group in which these activities were inadvertently not done.

Terminal Sacrifice:
On test day 7, all animals except the 6793 ppm exposure animals were anesthetized with carbon dioxide and euthanized. The 6793 ppm animals were kept for an additional week for further evaluation. A gross necropsy was not performed and tissues were not saved.

Results and discussion

Details on results:
Chamber Summary Data:
Actual average exposure concentrations were 246, 485, 542, 740, 1214, 3063 or 6793 ppm npropyl propionate. Airflow through the exposure chamber was maintained at 4 liters per minute for each exposure.

Respiration Rate Data:
A concentration-dependent decrease in respiration rate was observed. The mean maximum decrease in respiratory rate for each of the exposure groups were as follows: 30% (246 ppm), 46% (485 ppm), 52% (542 ppm), 60% (740 ppm) and 69% (1214 ppm).
Based on these data, the RD50 of inhaled n-propyl propionate was calculated to be 536 ppm, with a 95% confidence interval of 328-874 ppm.
The respiratory rate of each animal was measured during the ten minute period immediately following the end of exposure in order to assess the reversibility of the exposure-dependent decrease in respiratory rate. The percent recovery relative to the maximum respiratory rate depression for that animal was calculated for all animals in each experimental group. The average percent recovery for all experimental groups was 71.9% with a range of 50.1% (6793 ppm exposure group) to 82.8% (740 ppm exposure group).

Mortality:
Two animals died during the 6793 ppm exposure (one during the exposure to n-propyl propionate and one during the 10-minute recovery period), one animal died during the 3063 ppm exposure and one animal was found dead on the day following exposure to 1214 ppm. All other animals survived the 30 minute exposure to the test material as well as the one-week postexposure period.

Clinical Observations:
No abnormal in-life observations were noted post-exposure and all surviving mice appeared normal by test day two.

Body Weight:
With the exception of the mice in the 740 ppm exposure group, which gained weight throughout the study, the mean bodyweights for all groups decreased approximately 0.7-7.8% from test day one to test day two. Mean bodyweights for all exposure groups exceeded pre-exposure (test day one) values by the end of the seven-day observation period with the exception of the 6793 ppm exposure group.

Applicant's summary and conclusion

Conclusions:
Based on these data, the RD50 of inhaled n-propyl propionate is 536 ppm (95% CI = 328-874) for male Swiss Webster mice.
Executive summary:

This study was conducted to evaluate the sensory irritant potential of inhaled n-propyl propionate vapors in adult Swiss Webster (CFW) mice. The concentration of test material (n-propyl propionate) which resulted in a 50% depression in respiration rate (RD50) was determined according to procedures outlined in ASTM E 981-04 (Standard Test Method for Estimating Sensory Irritancy of Airborne Chemicals).

Groups of four male Swiss Webster (CFW) mice were exposed for thirty minutes to 246, 485, 542, 740, 1214, 3063 or 6793 ppm n-propyl propionate. Mice were allowed a tenminute

acclimation period before the exposure/monitoring cycle began. After the ten minute acclimation period the exposure/monitoring cycle was initiated and baseline respiratory rate data (breaths/minute) were collected for a ten-minute period. Test material exposure was then initiated and mice were exposed for 30 minutes with continuous collection of respiratory rate data. Respiratory rate during a ten-minute recovery period was recorded after the end of exposure.

The mice were held for seven days following exposure during which time body weights and clinical observations were monitored to assess any untoward effects of exposure, with the exception of the 542 ppm exposure group in which these activities were inadvertently not done.

Two animals died during the 6793 ppm exposure (one during the exposure to n-propyl propionate and one during the 10-minute recovery period), one animal died during the 3063 ppm exposure and one animal was found dead on the day following exposure to 1214 ppm. All other animals survived the 30 minute exposure to the test material as well as the one-week post-exposure period. No abnormal in-life observations were noted post-exposure and all surviving mice appeared normal by test day two.

With the exception of the 740 ppm exposure group, which gained weight throughout the study, mean bodyweights for all groups decreased approximately 0.7-7.8% from test day one to test day two. Mean bodyweights for all exposure groups exceeded pre-exposure (test day one) values by the end of the seven-day observation period with the exception of the 6793 ppm exposure group.

Based on these data, the RD50 of inhaled n-propyl propionate is 536 ppm (95% CI = 328- 874) for male Swiss Webster mice.