Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: inhalation
Remarks:
other: 10 day treatment on working days
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: for justification of read-across see endpoint study summary

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
10-day-inhalation study: Ten rats per sex per dose were exposed to the test compound (67, 200 and 670 mg/m³), or an air control (0 mg/m³) on 10 consecutive working days over a 14 day period in rats. Animals were evaluated based on clinical condition, body weight change, clnical pathology profiles and anatomical pathology assessments.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
structural analogue: see confidential details on test materials

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc., Kingston, New York
- Age at study initiation: 5-6 weeks
- Weight range at study initiation: males: 134.4 to 160.5 g; females: 109.0 to 122.8 g
- Fasting period before study: No data
- Housing: individually in stainless steel wire mesh cages, (manufactured by Lab Products, Inc., Maywood, New Jersey) which were located in a Hazleton 1000, whole-body, Inhalation exposure chamber.
- Diet (e.g. ad libitum): Certified Purina Rodent Chow® (pellets) was provided ad libitum during non-exposure periods
- Water (e.g. ad libitum): Water was provided ad libitum (except during the daily exposure periods)
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.44 - 25.00 °C
- Humidity (%): 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12 h /12 h

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: The count median aerodynamic diameters (CMAD) and the geometric standard deviations (GSD) were recorded from the APS results and the mass median aerodynamic diameters (MMAD) were calculated from the CMAD and GSD values using the Hatch-Choate equation. The overall mean values (all chambers combined, except air control) for the CMAD, MMAD, and GSD during the pre-animal exposure period were 0.72, 1.06, and 1.42, respectively. During animal study, CMAD ranged between 0.56 gm and 0.68 gm, and GSD values were between 1.22 and 1.41. The corresponding calculated MMAD ranged between 0.64 gm and 0.90 um.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Hazleton H1000, one cubic meter stainless steel and glass chambers were used for the test article and air control exposures.
- Method of holding animals in test chamber: The cages with the animales were located in a Hazleton 1000, whole-body, inhalation exposure chamber. There were 24 compartments (12 per side) in each cage unit. One cage unit was used to house all animals assigned to a particular exposure group for the study.
- System of generating particulates/aerosols:
• The test article generation system was designed to simulate a possible manufacturing process failure which could result in release of a pressurized stream of liquified structural analogue into the atmosphere of the work place, where likely exposure would be inhalation of vaporized structural analogue and/or condensation aerosols of the test article.
• This generation system was designed to produce the highest reasonably or likely achievable concentration of structural analogue generated from its liquid state and allowed to achieve room temperature equilibrium conditions.
• The primary component of the generation system was a Sonimist® spray nozzle (Model HS600-2, Heat Systems Ultrasonics, Inc., Farmingdale, New York). A fine stream of heated liquid test article was discharged at a variable rate at the outlet of this nozzle where it was dispersed by a jet of compressed air producing a sonic disturbance. The resulting spray of fine droplets vaporized. As the vapor cooled to room temperature, a fine condensation aerosol formed.
• The secondary component of the generation system consisted of a test chemical reservoir that was heated and pressurized for the delivery of the structural analogue to the Sonimist ® Nozzle. A 3 gallon galvanized canister was used as the reservoir and was modified to accept an internal core thermocouple and a manual pressure relief valve (in addition to the factory-installed automatic pressure relief valve). Both the reservoir and the liquid feed line were wrapped with heat tape to maintain the necessary temperature of the test article in a liquid state until it was vaporized. In addition, the carrier air line and the Sonimist ® nozzle were wrapped with heat tape. The temperature at several locations (reservoir core, reservoir skin, nozzle skin and carrier air line
skin) of the generation was controlled and measured.
• The Sonimist® nozzle discharged directly into a 1.3 cubic meter plenum chamber. This plenum chamber allowed the sonicated aerosol to vaporize and achieve equilibrium at room temperature.
- Exposure System
• The test article atmosphere within the plenum chamber was transported into a single manifold system that supplied the three animal exposure chambers.
• The total concentration of structural analogue aerosol in the plenum chamber and manifold air was approximately the same as that used for the high exposure level. The target concentrations for the 2 lower exposure levels were achieved by diverting a metered fraction of manifold air into the exposure chamber and diluting it with HEPA/activated-charcoal-filtered room air.
• Control rats were exposed in a chamber that received HEPA and activated charcoal-filtered room air only. The air handling system for the control chamber was separate from the test article generation and delivery system.
- Temperature, humidity, pressure in air chamber: 22.22 ± 2.78 °C, 55 ± 15 % recorded three times per day during the 1st, 3rd and 6th hour of animal exposure.
- Air flow rate and air change rate: Chamber air flow rates were controlled by orifice plate tube flowmeters and valves. Flow rates through the chambers were controlled to 15 ± 2 air changes per hour.
- Method of particle size determination:
Particles were assumed to be normally distributed with respect to the logarithm of their diameters for calculation of aerosol mass median aerodynamic diameters (MMAD) and geometric standard deviations (GSD). The aerodynamic diameters were defined as the diameter of unit density spheres which have the same settling velocity as particles of the test aerosol. Particle size distributions were measured and calculated using an APS 3310 Aerodynamic Particle Sizer with a 100:1 dilutor. During the pre-study development, particle size distributions were determined twice in each exposure chamber and twice in the plenum chamber. During the animal exposures, particle size distributions were measured once per exposure chamber (including air control) per day.
- Treatment of exhaust air: The exhaust air from all the chambers passed through Cambridge Sidelock® HEPA and Prefilter Assemblies (Cambridge Filter Corp., Syracuse, NY) to remove particulate structural analogue before combining the outflow into a Scrubber. The 'scrubbed' air was then vented outside the building.

TEST ATMOSPHERE
- Aerosol Concentration
• Because generation of structural analogue atmospheres under the test conditions produce both particulate and vapor phase emissions, a method was devised to sample both phases simultaneously. A calibrated rotameter and flow controller was used to draw an air sample from each chamber or sample location. The sample was first drawn through a 25 mm glass fiber filter mounted in an openfaced Delrin filter holder attached to a sample line inserted into the chamber near the breathing zone of the animals. The glass fiber filtered the particulate material while the vapor phase material remained entrained
in the sampling air stream and was directed into a Infrared Analyzer where the total vapor concentration was measured.
• The sample flow rate was set at 5 liter per minute while sample duration varied according to chamber test article concentration.
• Gravimetric mass was calculated from the filter weight gain and the sample volume.
• Vapor phase concentration was determined by measuring the absorbance of each sample and mathematically regressing the absorbance value from a calibration curve to calculate the mass vapor concentration.
• The two values, gravimetric and vapor concentrations, were combined to give the total mass concentration of structural analogue and were also expressed as the percent of total mass.
The Miran 1A Infrared Analyzer was equipped with a 20 meter variable pathlength gas sample cell. A standard calibration loop consisting of an injection port and circulation pump was added for calibration purposes. In this way, by knowing the volume of the sample cell and the volume of the injected amount of test material, the concentration in the cell was calculated. The absorbance (or in this case the analog output in VDC) was observed for a given injection or calibration point. The data were plotted and regressed such that input at an absorbance valve was derived for each known mass concentration value.
- Calibration
• The Miran 1A was calibrated during the pre-study development. First, a suitable wavelength was selected by completing a full infrared spectrum scan of structural analogue. Because of the physical properties of structural analogue at room temperature (solid phase), preventing injection of the test compound in liquid form, the structural analogue was dissolved in reagent grade methanol. A methanol scan was also performed. By comparing the scans, a wavelength of 8.25 gm at a detection cell pathlength of 20.25 meters was selected to be the most specific for structural analogue without interference from methanol and water vapor.
• Using known amounts of structural analogue standard prepared in methanol the absorbance value were recorded. A standard was prepared in reagent grade ethanol at a concentration of 0.204502 g/mL. A series of injections into the Miran closed-loop calibration system were conducted in triplicate. The Miran detection cell was maintained at 70°C to prevent condensation of structural analogue. The calibration was conducted over a concentration range of 4 ppm to 167 ppm (18 mg/m³ to 724 mg/m³).
- Samples taken: During the animal studies, all locations (as described above) were sampled once per hour with the gravimetric aerosol/Miran vapor sampling system, except that gravimetric samples were not collected from the room air, the air control chamber, or the Mystaire ® scrubber exhaust.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas chromatography (GC) of impinger samples was the chosen technique. One hundred twenty-five mL gas washing bottles were filled with 100 mL of reagent grade methanol. Two impingers in series were placed into a wet ice bath and a 5 liter per minute sample was drawn from the exposure chamber. Simultaneous samples were collected with the gravimetric/Miran system. After the completion of the impinger sampling, the sample lines were backflushed with methanol to retrieve condensed material. The samples were reconstituted to 100 mL volume and analyzed directly using a gas chromatograph.
For the GC a 30 m x 0.75 mm Supelco SPB-1 column with a 1.5 micron film thickness was used. The oven was set at 70°C with the injector and detector at 90°C. The carrier flow was at 20 mL/min. Injection volumes were constant at 0.6 L. To establish a standard calibration curve, feference test material was dissolved in reagent grade methanol between the ranges of 25 gg/mL to 764 gg/mL. The correlation coefficient for the curve was 0.999.
Actual impinger samples were obtained from each exposure level during the second exposure day of the animal study to verify the results of the gravimetric/Miran atmosphere measurement system. The results show that there was good agreement between gravimetric/vapor determination and GC determination methods.
Duration of treatment / exposure:
14 days
Frequency of treatment:
6 hours/day, 5 days/week for a total of 10 exposures
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 67, 200, 670 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 84.4, 227, and 731 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
other: filtered air alone
Details on study design:
- Dose selection rationale: The exposure concentrations for the study were selected to simulate, at the highest level, the theoretical worst-case exposure condition; with lower concentration values chosen to evaluate the concentration response. The Inhalation exposure system was designed to simulate release of heated, liquified structural analogue into ambient atmosphere and to deliver a near-saturated atmosphere of respirable test article to the study animals. Preliminary studies showed that 670 mg/m³ represented the approximate-saturation concentration of the respirable fraction (comprised of both vapor and aerosol phases) of structural analogue in absence of air under dynamic air-flow conditions. The middle and low concentrations represented approximated half-log interval decreases from the maximum achievable concentration. The lowest concentration level examined, was at least 2 to 3 orders of magnitude greater than the average concentration of the test article measured in the workplace.
- Post-exposure recovery period in satellite groups: None, animals were killed one day after the last exposure.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes,
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes,
- Time schedule: twice daily (in the morning, at least 6 hours apart, before 10:00 a.m. and after 2:00 p.m., 7 days per week) before and after each daily exposure or at similar intervals on nonexposure days for clinical evidence of toxicity or other abnormalities.
The clinical evaluations included a thorough examination of the animals general condition, exterior appearance, external orifices and observable mucous membranes. The animals were handled and allowed to move to evaluate general behavior, coordination and general neuro-muscular function. All
observations were recorded on a Data Management System.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight values of all animals were recorded on Study Day -8 (within 48 hours of receipt) and again on Study Day -2 for randomization and assignment to study groups. The body weights of animals assigned to the study were determined on Study Day 1, prior to the first exposure and again on Study Days 8, 14, and prior to scheduled necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, from the retro-orbital plexus
- Time schedule for collection of blood: shortly before necropsy
- Anaesthetic used for blood collection: Yes (identity), propylene glycolfree sodium pentobarbital
- Animals fasted: yes, approximately 12 hours prior to necropsy, water was provided ad libitum until the time of necropsy
- How many animals: all animals
- The following parameters were examined:
• red blood cell count (RBC, 10e6 cells per microliter)
• hematocrit (HCT, percent)
• hemoglobin (Hgb, g/dL)
• mean corpuscular volume (MCV, femtoliters)
• mean corpuscular hemoglobin (MCH, picograms)
• mean corpuscular hemoglobin concentration (MCHC, percent)
• white blood cell count (WBC, 10³ per microliter)
• platelet count (PLAT, 103 platelets per microliter)
Smears for differential cell counts were also made from the blood samples and were stained using a modified Wright-Giemsa stain.
• The relative number of nonsegmented neutrophils (BAN),
• segmented neutrophils (SEG),
• eosinophils (EOS),
• basophils (BAS),
• lymphocytes (LYM), and
• monocytes (MON) was determined for each animal.
• The absolute number of each cell type per microliter was also calculated.
• The number of nucleated red blood cells per 100 white blood cell (nRBC/100 WBC) was also determined.
• The total reticulocyte count (RETIC) was determined after red blood cells were pre-stained with new methylene blue and a smear prepared.
• The number of reticulocytes per 100 red blood cells was counted microscopically and the results reported as the percentage of red cells which were
reticulocytes.
CLINICAL CHEMISTRY: Yes, from the retro-orbital Sinus
- Time schedule for collection of blood: shortly before necropsy
- Animals fasted: yes, approximately 12 hours prior to necropsy, water was provided ad libitum until the time of necropsy
- How many animals: All animals
- The following parameters were examined:
• blond urea nitrogen (BUN, mg/dL)
• creatinine (CRET, mg/dL)
• fasting glucose (GLU, mg/dL)
• total protein (TP, g/dL)
• albumin (ALB, g/dL)
• globulin (GLO, A:G ratio)
• cholesterol (CHOL, mg/dL)
• lactate dehydrogenase (LDH, IU/L)
• serum alanine aminotransferase (ALT, IU/L)
• serum aspartate aminotransferase (AST, IU/L)
• alkaline phosphatase (ALP, IU/L)
• bilirubin (Bil, mg/dL)
• calcium (CA, meg/L)
• sodium (NA, meg/L)
• potassium (K, meg/L)
• chloride (CL, meg/L)
• phosphorus (IP, mg/dL)
URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes,
- Each animal was killed by exsanguination
- Complete gross examination, with special attention given to the lungs and upper respiratory tract.
- Organs weighed from all animals were: liver, lung, kidneys (pair), and heart (excluding major vessel).
- Organ/body weight ratios were calculated.

HISTOPATHOLOGY: Yes, liver, kidney, heart, mainstem bronchi, lung trachea, nasal cavity
- The respiratory tract, defined as the lungs, nasal cavity (four sections), nasopharynx, larynx (two cross-sections), and trachea (crossand longitudinal sections), and all gross lesions suspected to be exposure-related, from all animals of each exposure group were blocked for slides, sectioned, stained with hematoxylin and eosin (H&E), and submitted for light microscopic examination. The lungs were sectioned so as to present a maximal section of the mainstem bronchi.
- The nasal cavity was prepared in four sections using the landmarks described by Young (Fundam. Appl. Toxicol., 1: 309-312, 1981).
- All other tissues collected from all other animals were preserved and held in formalin for possible future histopathological evaluation.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
- All animals survived to study termination.
- Clinical signs of toxicity were confined to male and female rats exposed to the high-concentration (670 mg/m³) of structural analogue. A red nasal discharge was observed in all animals in the high-concentration group, beginning on Study Day 1. This condition was apparent at the end of the 6-hour exposure period and would abate overnight, with the animals appearing nearly normal the next morning.
- Nasal discharge, red: Males: day 1: 10/10, day 2 : 2/10, day 4 - 8: 10/10 and day 12 - 14: 10/10; Females: day 1: 10/10, day 4 - 8: 10/10 and day 12 - 14: 10/10;
- There were no other clinical or behavioral changes observed in the high concentration group that were thought to be related to structural analogue exposure.
- In addition, all other rats at the middle and low exposure concentration group were similar to control animals in behavior and in general health and clinical appearance.

BODY WEIGHT AND WEIGHT GAIN
- At randomization and on Study Day 1, all exposure groups had group mean body weights that were statistically similar to that of their respective control group. The 200 and 670 mg/m³ male exposure groups has group mean body weight values that were significantly decreased relative to control; -5.6 % and -10.2 %, respectively on Study Day 8 and -6.2 % and -13.1 %, respectively on Study Day 14.
- The 670 mg/m³ female group had a group mean body weight value that was significantly less than the female control group (-8.8 % on Study Day 8 and -11.2 % on Study Day 14).
- No clinical signs indicative of emaciation or thinness were apparent in animals showing decreased weight gains relative to those of control animals.
- All other male and female groups had group mean body weight values that were similar (p > 0.05) to those of their respective control group.

- The terminal body weights of male rats exposed to 200 and 670 mg/m³ of structural analogue were significantly less (p < 0.05 and 0.01, respectively) than the control group (after 8 days: control: 178.8 g; 200 mg/m³: 168.7 (5.6 %) and 670 mg/m³: 160.8 g (10.1 %); after 14 days: control: 185.4 g; 200 mg/m³: 173.9 (6.2 %) and 670 mg/m³: 181.2 g (2.3 %)). The group mean terminal body weights of females (the 670 mg/m³ exposure group) were also significantly decreased (p < 0.01) compared to control animals (after 8 days: control: 131.7 g; 670 mg/m³: 120.1 g (8.8 %); after 14 days: control: 132.3 g; 670 mg/m³: 117.5 g (11.2 %)).
FOOD CONSUMPTION
- no data

FOOD EFFICIENCY
- no data

WATER CONSUMPTION
- no data

OPHTHALMOSCOPIC EXAMINATION
- no data

HAEMATOLOGY
- Platelet Count was significantly changed in males of the mid and high dose group: control: 749.70 x 10³/µL; 200 mg/m³: 646.90 x 10³/µL; 670 mg/m³: 704.90 x 10³/µL.
- Hemoglobin was significantly changed in males of the high dose group: control: 16.71 g/dL; 200 mg/m³: 16.78 g/dL; 670 mg/m³: 16.19 g/dL.
- WBC Differential Count / Monocyte was significantly changed in females of the mid dose group: control: 0.70 x 10³/µL; 200 mg/m³: 1.80 x 10³/µL; 670 mg/m³: 1.11 x 10³/µL.
- These changes were small in magnitude and not considered to be related to structural analogue exposure.

CLINICAL CHEMISTRY
While there were some statistically significant changes (Creatinine: 670 mg/m³ females (control: 0.63 mg/dL; 670 mg/m³: 0.556 mg/dL); Total Protein: 200 mg/m³ females (control: 6.48 g/dL; 67 mg/m³: 6.22 g/dL); Albumin 67 mg/m³ females (control: 4.43 g/dL; 67 mg/m³: 4.28 g/dL); Globulin 670 mg/m³ males (control: 2.10; 670 mg/m³: 1.94); A/G Ratio 670 mg/m³ males (control: 2.11; 670 mg/m³: 2.36); Alanine Aminotransferase 670 mg/m³ females (control: 41.90 IU/L; 670 mg/m³: 53.44 IU/L); Alkaline Phosphatase 670 mg/m³ males (control: 413.80 IU/L; 670 mg/m³: 363.30 IU/L)), these changes were small in magnitude, were not concentration-dependent and were within the normal physiological ranges for this strain of rats. Therefore, none of the variations in clinical pathology parameters were considered to be due to structural analogue exposure.

URINALYSIS
- no data

NEUROBEHAVIOUR
- no data

ORGAN WEIGHTS
• Kidney: Absolute kidney weight value of male (7.5 %) and female (12.4 %) rats in the 670 mg/m³ group were significantly greater (p < 0.05 and 0.01, respectively) than control animals. Kidney to body weight ratios were also significantly increased (p < 0.01) in the male (22.6 %) and female (25.4 %) 670 mg/m³ exposure groups.
• Heart: There were no significant differences between the exposure and control animals in the absolute mean heart weights. However, there was a significant increase (p < 0.01) in organ-to-body weight ratios for the male (11.8 %) and female (9.2 %) high exposure (670 mg/m³ ) groups
compared to control animals.
• Lung: There were no significant differences (p > 0.05) between the exposure and control groups in absolute mean lung weights.
However, the female high concentration (670 mg/m³) group, had significantly elevated (p < 0.05) lung-to-body weight ratios (9.5 %) relative to control rats.
• Liver: There were no significant differences in group mean absolute liver weights between any groups. However, the male (21.8%) and female (12.4 %) high-concentration (670 mg/m³) groups had significantly increased liver to- body weight ratios.

GROSS PATHOLOGY
- There were few and only incidental gross tissue changes present in the study animals. The gross lesions that occurred were sporadic and were not considered exposure related. These lesions were common spontaneous lesions that were expected to occur in a group of Fischer 344 rats of this age range. These lesions consisted of an apparent cystic left ovary in one air control female, and 3 low-concentration group females, and a red, discolored mediastinal lymph node in one middle-concentration (200 mg/m³) male.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Lesions considered to be related to the inhalation exposure of structural analogue were confined to the nasal cavity of the high-concentration group animals of both sexes and were present in the same anatomical location and at essentially the same degree of severity from both sexes.
- These changes were identified in levels II, III, and IV of the nasal cavity with level IV showing the most extensive changes. Specifically, these lesions involved the olfactory epithelium lining the dorsal meatus, dorsal portion of the nasal septum, and the adjacent ethmoid turbinates.
- Morphologically, the lesions consisted of necrosis/degeneration of the affected olfactory epithelium. This was most severe and extensive in the area of the dorsal meatus and nasal septum. The lesion was more focal to multifocal where it involved the epithelium covering the adjacent ethmoid turbinates. - In conjunction with the olfactory epithelial necrosis/degeneration was a moderate degree of a serofibrinous/suppurative inflammation which was also most severe and extensive in the immediate area of the dorsal meatus. The overall thickness of the affected regions of olfactory epithelium decreased in proportion to the amount of involvement of the inflammatory, necrotic and degenerative lesions and ranged from a slight change in the less affected regions, to a gradual thinning into a single layer of cuboidal cells, and finally to complete denudation in the most severely affected areas involving the dorsal meatus.
- In some areas, the affected mucosa had changes consistent with a diagnosis of squamous metaplasia. The submucosa in the region of the dorsal meatus was thickened, secondary to the presence of inflammatory exudate, edema fluid, and proliferating loose fibrous connective tissue.
- In many areas the involved olfactory epithelium had inverted or became trapped in the underlying submucosa where it formed pseudorosettes or pseudoacinar/glandular structures which were lined by columnar, cuboidal, or squamoid appearing olfactory epithelium. Many of these structures were ectatic to varying degrees and contained inflammatory debris and proteinaceous fluid within the Lumen. Six of the high-concentration group females
and three of the high- concentration group males (670 mg/m³) had bilateral adhesions between the adjacent ethmoid turbinates and the dorsal portion of the nasal septum and/or the wall of the dorsal meatus. These adhesions were becoming organized through the proliferation of dense fibrous connective tissue and most probably would have remained as a permanent malformation in the nasal cavity if exposure was discontinued and resolution of the
necroinflammatory process was allowed to take place.
- Although not diagnosed separately, olfactory nerve fibers were occasionally noted to be somewhat vacuolated in appearance, usually in association with areas of significant inflammation. Whether this represented fixation artifact, or early signs of neural degeneration, either as a direct result of exposure to the test article or secondarily as a result of the inflammation, could not be definitively ascertained.
- Exposure-related lesions involving the nasal cavity in the high-concentration group (670 mg/m³) male and female rats are summarized in the following
table:
• Moderate, necrosis/degeneration of the olfactory epithelium: Males: 10/10, Females: 10/10
• Moderate, suppurative, inflammation of the olfactory epithelium: Males: 10/10, Females: 10/10
• Moderate, squamous, metaplasia, of the olfactory epithelium. Males: 10/10, Females: 10/10
• Adhesions, of the turbinate-dorsal wall: Males: 1/10, Females: 1/10
• Adhesions, of the turbinate-septum: Males: 2/10, Females: 5/10
- After examining the respiratory tract tissues from the high concentration and air-control rats, these same tissue sections collected from the low- and mid-concentration rats of both sexes were trimmed, processed, stained with hematoxylin and eosin and examined microscopically in an attempt to establish a no effect level (NOEL) for this particular compound. All tissues required to be examined histopathologically were present from the low-and middle-concentration rats of both sexes with the exception of the larynx from one middle-concentration (200 mg/m³) female rat, which was reported to be missing at trim. Histopathologically, the lesions involving the nasal cavities seen in the high-concentration animals were not present in the low- and middle-concentration rats of both sexes.
- Therefore, the NOEL for this spectrum of lesion in rats of both sexes under the exposure conditions used in this study was determined to be 200 mg/m³.
- Small, focal, subpleural aggregations of lymphocytes/macrophages were also seen in the lungs of treated rats of both sexes. However, these changes were also seen in control male and female rats and therefore, were considered to be an incidental finding and not related to the exposure to structural analogue. Other than these small pulmonary inflammatory foci, no other lesions involving the lungs or the rest of the lower respiratory tract (nasopharynx, larynx, trachea) were observed in control or exposed rats of either sex.
- In conclusion, the whole-body inhalation exposure of rats to structural analogue for 10 days at a concentration of 670 mg/m³ resulted in a moderate degree of necrosis/degeneration of the olfactory epithelium lining of the nasal cavity in areas covering the dorsal meatus, adjacent nasal septum, and nasal turbinates. Associated changes consisted of a serofibrinous/suppurative inflammatory response, squamous metaplasia of affected epithelium and adhesions between the ethmoid turbinates and the nasal septum or the wall of the dorsal meatus. The lesions were of approximately the same extent and severity in both sexes with the exception of the adhesions which were more prevalent in the high-concentration group female rats. Microscopic lesions were identified in levels II, III, and IV of the nasal cavity with those involving level IV being the most extensive.
- Histopathologic examination of the respiratory tract tissues collected from the lower concentration group rats of both sexes did not reveal the nasal lesions seen in the high-concentration group rats. No changes in the lower respiratory tract (nasopharynx, larynx, trachea, and lungs) considered to be related to the inhalation of structural analogue were found. These irritation-related effects, seen only at the highest exposure level, were not unexpected for a respiratory irritant material, and could be explored further through additional evaluations, if warranted. A NOEC for this effect was determined to be 200 mg/m³.

Effect levels

open allclose all
Dose descriptor:
NOEC
Remarks:
local
Effect level:
>= 200 mg/m³ air
Sex:
male/female
Basis for effect level:
other: At highest dose, 670 mg/m3, microscopic findings included necrosis/degeneration, inflammation, and squamous cell metaplasia of the olfactory epithelium and nasal septum adhesions.
Dose descriptor:
NOEC
Remarks:
systemic
Effect level:
>= 200 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Decreased group mean body weight values (18 %).
Dose descriptor:
LOEC
Remarks:
local and systemic
Effect level:
670 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Decreased group mean body weight values (18%); red nasal discharges; necrosis/degeneration, inflammation, and squamous metaplasia of the olfactory epithelium and nasal septum adhesions.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The test atmosphere generation and delivery system used in this study functioned well and as designed. Heated, pressurized liquid structural analogue was delivered into a plenum of air held at room temperature, where the material vaporized and condensed, achieving equilibrium (under dynamic air flow conditions). The atmosphere within the plenum was effectively saturated at the nominal air flow of 350 L/min used to entrain and deliver the test atmosphere. The liquid structural analogue delivered to the plenum stabilized into a biphasic atmosphere, consisting of vapor and a fine condensation solid aerosol. The percentage of the total mass concentration present in aerosol phase within the animal exposure chambers was directly proportional to the total structural analogue concentration. Therefore, as portions of the saturated manifold atmosphere were diluted in the middle- and low-concentration chambers, a lower percentage of the chamber atmosphere was in particle phase.

Observation of toxic effects during this repeated exposure inhalation study was generally limited to the highest exposure level of structural analogue. Male and female rats exposed to 670 mg/m³ structural analogue had decreased group mean body weight values relative to controls of approximately 10 percent. The reduced body weights were apparent on Study Day 8 and continued until study termination. Rats exposed to 670 mg/m³ also showed red nasal discharges that were apparent after the end of each daily exposure, which was indicative of upper respiratory tract irritation and ulceration. Microscopic examinations showed that rats exposed to 670 mg/m³ of structural analogue had necrosis/degeneration, inflammation, and squamous metaplasia of the olfactory epithelium and nasal septum adhesions. Squamous cell metaplasia is a dedifferentiation change that may become progressive and more proliferative in nature with continued exposure and is often considered to be a pre-neoplastic lesion.

While male and female rats exposed at the 670 mg/m³ level showed significant increases in absolute kidney weight relative to control and increases in several organ-to-body weight ratios, the toxicological significance of these findings is unclear in the absence of clinical chemistry and microscopic evaluations. Male rats exposed to 200 mg/m³ structural analogue had smaller body weight decreases of about 6 percent on Study Day 8.

No clinical signs of toxicity or treatment-related microscopic findings were seen in rats exposed to 67 or 200 ppm structural analogue. There were no concentration-related changes in clinical pathology studies that were considered to be biologically significant or exposure-related.

The observed lesions are considered to be typical of a respiratory irritant and are not unexpected following exposure to such a compound. The changes were noted only after repeated exposure to the highest concentration (670 mg/m³) used in this study, which would classify this test material as a mild to moderate acute respiratory irritant.

In conclusion, based on the results of this study the no-observable adverse effect concentration for structural analogue following repeated, 6 -hour exposures is 200 mg/m³.

Applicant's summary and conclusion