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Repeated dose toxicity: inhalation

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short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 16, 1989 to May 7, 1991
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented non-guideline study performed under GLP.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
14 day inhalation study with exposure 5 days/week.
GLP compliance:
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): 2,6-xylenol
- Physical state: white solid at room temperature (melting point 49 degrees C)
- Analytical purity: 100 %
- Storage condition of test material: controlled access at room temperature

Test animals

Fischer 344
Details on test animals and environmental conditions:
- Source: Charles River Breeding Laboratory, Inc., Kingston, NY
- Age at study initiation: 5-6 weeks (at receipt)
- Weight at study initiation: 134.4-160.5 g (males at randomization); 109.0-122.8 g (females at randomization)
- Housing: Animals were individually housed in stainless steel wire mesh cages which were located in a Hazleton 1000, whole-body, inhalation exposure chamber. One cage unit was used to house all animals assigned to a particular exposure group.
- Diet: ad libitum (during non-exposure periods)
- Water: ad libitum
- Acclimation period: 10 days

- Temperature (°C): 67-77 degrees F
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: June 16, 1989 To: June 30, 1989

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Remarks on MMAD:
MMAD / GSD: Pre-animal exposure characterization: The overall mean values (all chambers combined, except air control) for the CMAD, MMAD, and GSD were 0.72, 1.06 and 1.42, respectively.
System performance during study: Count median aerodynamic diameters ranged between 0.56 um and 0.68 um, and GSD values were between 1.22 and 1.41. The corresponding calculated mass median aerodynamic diameters ranged between 0.64 um and 0.90 um.
Details on inhalation exposure:
- Exposure apparatus: Hazleton H1000, one cubic meter stainless steel and glass chambers were used.
- Method of holding animals in test chamber: Animal cages were housed within the exposure chambers.
- System of generating particulates/aerosols: The primary component of the generation system was a Sonimist spray nozzle. A fine stream of heated liquid test substance was discharged at a variable rate at the outlet of this nozzle where it was dispersed by a jet of pressed air producing 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 substance reservoir that was heated and pressurized for the delivery of the test substance to the Sonimist Nozzle. A 3 gallon galvanized canister was used as the reservoir and was modified to accept an internal core thermocouple and 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 substance 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 of the generation was controlled by variacs and was measured by type 'T' thermocouples at the following locations: reservoir core, reservoir skin, nozzle skin and carrier air line skin. 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.
The test substance atmosphere within the plenum chamber was transported into a single manifold system that supplied the three exposure chambers. The total concentration of test substance 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.
- Temperature, humidity, pressure in air chamber: 72 +/- 5 degrees F; 55 +/- 15 % humidity
- Air flow rate: 15+/-2
- Method of particle size determination: 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 exposure, particle size distributions were measured once per exposure chamber per day.
- Treatment of exhaust air: Exhaust air passed through Cambridge Sidlock HEPA and Prefilter Assemblies to remove particulate before combining the outflow into a Mystaire scrubber utilizing a 50:50 mixture of chlorine bleach and water. The scrubbed air was then vented outside the building.

- Brief description of analytical method used: Because generation of test substance atmospheres produced both particulate and vapor phase emissions, a method was devised to sample both phases simultaneously. A calibrated rotameter and flow control was used to draw air samples from each chamber. These samples were first drawn through a 25 mm glassfiber filter mounted in an open faced 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 in the sampling air stream and was directed into a Miran 1A Infrared Analyzer where the total vapor concentration was measured. The sample flow rate was set at 5 L per minute while sample duration varied according to chamber test substance 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.
During the pre-study development, techniques were defined to verify the chamber test substance concentrations by chemical analysis. Gas chromatography of impinger samples was the chosen technique. One hundred twenty five mL gas washing bottle with glass fronts 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 back flushed with methanol to retrieve condensed material. The samples were reconstituted to 100 mL volume and analyzed directly using a gas chromatograph. A Varian Model 6000 gas chromatography was used with 30m x 0.75mm Supelco SPB-1 column with a 1.5 micron film thickness. The oven was set at 70 degrees C with the injector and detector at 90 degrees C. The carrier flow was at 20 mL/min. Injection volumes were constant at ~0.6 uL. 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.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Pre-animal exposure characterization: The total exposure chamber mass concentrations were within an average of 16.2% of target values and reproducible on a daily basis, with a <25% RSD. The mean concentration values for the 67, 200 and 670 mg/m3 groups were 84.4, 227 and 731, respectively, averaged over the three separate 6-hour trial generation periods. The greatest RSD was 22.6%, measured in the 200 mg/m3 chamber, while the greatest deviation from target concentration values was 26% which occurred in the 67 mg/m3.
System performance during study: All mean total concentration values had relative standard deviations of less than 6 %. Deviations from targeted concentrations were all less than or equal to 5 %. Within each chamber all mean vapor phase concentrations had RDSs of less than 6%. Results showed there was good agreement between gravimetric/vapor determination and GC determination methods. The largest variance between the two was 17%.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
Animals were treated 5 days/week for a total of 10 exposures, conducted within 14 days.
Doses / concentrations
Doses / Concentrations:
67, 200 and 670 mg/m3
nominal conc.
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Concentrations were selected to simulate, at the highest level, the theoretical worst-case exposure condition; with lower concentration values chosen to evaluate the concentration response.


Observations and examinations performed and frequency:
- Time schedule: Animals were observed twice daily for clinical evidence of toxicity or other abnormalities. Evaluations included a thorough examination of the animal’s general condition, exterior appearance, external orifices and observable mucous membranes. The animals were handled and allowed to move to evaluate general behaviour, coordination and general neuro-muscular function.

- Time schedule for examinations: Study Day 1, prior to the first exposure, and again on Study Days 8, 14 and prior to necropsy.

- Time schedule for collection of blood: shortly before necropsy
- Anaesthetic used for blood collection: Yes; propylene glycol-free sodium pentobarbital
- Animals fasted: yes
- How many animals: all
- Parameters examined: Each blood samples was analyzed for the following: red blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cell count, and platelet count. Smears for differential cells counts were made. The absolute number of each cell type per microliter was calculated. The number of nucleated red blood cells per 100 white blood cell was determined. The total reticulocyte count was determined after red blood cells were pre-stained and a smear prepared. In addition, the number of reticulocytes per 100 red blood cells was counted.

- Time schedule for collection of blood: shortly before necropsy
- Animals fasted: yes
- How many animals: all
- Parameters examined: Each blood sample was analyzed for the following: blood urea nitrogen, creatinine, fasting glucose, total protein, albumin, globulin, cholesterol, lactate dehydrogenase, serum alanine aminotransferase, serum aspartate aminotransferase, alkaline phosphatase, bilirubin, calcium, sodium, potassium, chloride, and phosphorus.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes; Each animal was given a 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. The following tissues were carefully examined, dissected from the carcass and preserved: liver, ling, kidney, trachea, heart, nasal cavity and mainstem bronchi.
HISTOPATHOLOGY: Yes; The respiratory tract, defined as the lungs, nasal cavity (four sections), nasopharynx, larynx (2 cross-sections), and trachea (cross and longitudinal sections) and all gross lesions suspected to be exposure related were examined. The lungs were sectioned so as to present a maximal section of the mainstem bronchi.
Normally distributed data (parametric) was analyzed for treatment effects by analysis of variance and pairwise comparisons made between groups using Dunnett’s multiple range t-test. Non-parametric data was analyzed by the Kruskal Wallis test and by the Mann-Whitney U Test for pairwise group comparisons.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY-Clinical signs of toxicity were confined to male and female rats in the high dose group. A red nasal discharge was observed in all animals in the high dose 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. This was noted by the authors to be a sign of upper respiratory tract irritation and ulceration. No other findings were observed.

BODY WEIGHT AND WEIGHT GAIN-The mid and high dose males had 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 high dose females 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.
The terminal body weights of males in the mid and high dose groups were significantly less (p
HAEMATOLOGY- While there were some statistically significant changes in the measured parameters, these changes were small in magnitude, were not concentration-dependent and were within the normal physiological ranges for this strain of rat. Therefore, none of the variations were considered to be test substance related.

CLINICAL CHEMISTRY- While there were some statistically significant changes in the measured parameters, these changes were small in magnitude, were not concentration-dependent and were within the normal physiological ranges for this strain of rat. Therefore, none of the variations were considered to be test substance related.

ORGAN WEIGHTS- Absolute kidney weight value of male and female rats in the high dose group were significantly greater (pThere was a significant increase (pThe high dose female group had a significantly elevated (pThe male and female high dose 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.

HISTOPATHOLOGY: NON-NEOPLASTIC-Lesions considered to be related to the treatment were confined to the nasal cavity of the high dose males and females and were present in the same anatomical location and at essentially the same degree of severity from both sexes. These changes were indentified 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 lesions were 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 exudates, 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 protein aceous fluid within the lumen. Six of the high dose females and three of the high dose males 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 treatment or secondarily as a result of the inflammation, could not be definitely ascertained.
After examining the respiratory tract tissues from the high dose and air control rats, these same tissue sections collected from the low and mid dose rats of both sexes were trimmed, processed, stained and examined in an attempt to establish a NOEL. All tissues required to be examined histopathologically were present from the low and mid dose rats with the exception of the larynx from one mid dose female, which was reported to be missing at trim. Histopathologically, the lesions involving the nasal cavities seen in the high dose animals were not present in the low and mid dose rats. Therefore, the NOEL for this spectrum of lesion in rats of both sexes under the exposure conditions used 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 treatment. Other than these small pulmonary inflammatory foci, no other lesions involving the lungs or the rest of the lower respiratory tract were observed.

Effect levels

open allclose all
Dose descriptor:
Effect level:
200 mg/m³ air (nominal)
Based on:
test mat.
Dose descriptor:
Effect level:
670 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: Based on reduced body weight and red nasal discharge upon exposure which was deemed indicative of upper respiratory tract irritation and ulceration. This was supported by histopathology.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Male and female rats were treated with 2,6-xylenol (67, 200, 670 mg/m3) by whole body inhalation for 6 hours/day, 5 days/week, for a total of ten exposure, conducted within 14 days. The NOAEC was determined to be 200 mg/m3. The LOEC was determined to be 670 mg/m3.