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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-11-15 to 1994-12-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium nitride
EC Number:
246-140-8
EC Name:
Aluminium nitride
Cas Number:
24304-00-5
Molecular formula:
AlN
IUPAC Name:
alumanylidyneamine
Test material form:
aerosol dispenser: not specified
Remarks:
migrated information: aerosol
Details on test material:
- Name of test material (as cited in study report): aluminum nitride
- Molecular weight (if other than submission substance): 41.0 daltons
- Impurities (identity and concentrations): aluminum oxide (0.5%)
- Lot/batch No.: 931004-DT: a blend of DOW AlN (PL44RB19) and Tokuyama soda AlN (TS36250)
- Storage condition of test material: Packaged individuaⅡy to minimize exposure ofthe samples to water vapor by repeatedly opening one bottle

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Kingston, NY.
- Age at study initiation: 8 weeks
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002, ad libitum (except during exposure)
- Water (e.g. ad libitum): municipal tap water, ad libitum (except during exposure)
- Acclimation period: one week (subsequently acclimated eight times over approximately two weeks to periods of nose-only exposure device restraint)

ENVIRONMENTAL CONDITIONS
- Animals were placed in rooms designed to maintain adequate environmental conditions concerning temperature, relative humidity and photocycle for the specific species under test.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
not specified
Remarks on MMAD:
MMAD / GSD: MMAD: 1.5 um
GSD: 1.6
84% of the AlN aerosol mass in particles less than 2.4 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed to test material in ADG or ADG type nose-only chambers with ADG restrainer systems (ADG instruments, Inc., Codicote, England) under dynamic air flow conditions.
- System of generating particulates/aerosols: Aerosols of aluminum nitride were generated with a particulate aerosol generator. A stainless steel cup held approx. 50 grams of AIN, and a screw driven bladed cap was driven into the cup. Air ports in the bladed cap swept the test material into the airstream of the aerosol distribution system.
- Temperature, humidity, pressure in air chamber: 22 °C, humidity 40-60%, neutral pressure
- Air flow rate: 30 liters/minute, determined with a manometer
- Method of particle size determination: particle size distribution in each chamber was determined (at least) weekly with a six-stage cascade impactor (Model 266, Sierra Instruments, Carmel, CA).
- Treatment of exhaust air: A distributor exhaust line was used which vents AlN aerosol to a high efficiency filter. The air flow in this exhaust line was not determined and, therefore, the usage cannot be precisely related to chamber "nominal" concentration.

TEST ATMOSPHERE
- Brief description of analytical method used: The mass concentration of test material in the chamber was monitored with weighed filter samples, at least four samples per chamber per day (TE 36 filters, 0.45 µm pore size, Schleicher and Schuell, Keene, NH). A constant volume pump (Model 110, Sierra Instruments, Carmel, CA) was used to collect air samples for filters and cascade impactors at 3 liters/min
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The mass concentration of test material in the chamber was monitoted with weighed filter samples, at least four samples per chamber per day
- Constant volume pump was used to collect air samples for filters and cascade impactors at 3 liters/min
- A TSI APS 33 laser velocimeter was used to help monitor the AIN concentration in the distribution system
Duration of treatment / exposure:
19 exposures during a 4-week study period
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2, 12, and 60 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 2, 10, and 70 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes
Details on study design:
- Necropsies were conducted on five rats/sex/dose on the day after the last exposure. The remaining 5 rats/sex/dose were the satellite group and were housed for additional 10 weeks without additional exposure. Afterwards, necropsies were performed and tissues saved.
Positive control:
N.A.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: overt signs of toxicity and changes in demeanor

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of the study and weekly thereafter
- Observations: evaluation of the skin and fur, eyes, mucous membranes, respiration nervous system and behaviour pattern. Observations for lethargy, tremors, convulsions, salivation, lacrimation, diarrhea and other signs/of altered central nervous system function

BODY WEIGHT: Yes
- Time schedule for examinations: on test days 1, 3, 5 ,8, 11 and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the initial exposure.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prioir to necropsy (at week 4 and 10)
- Anaesthetic used for blood collection: Yes, methoxyfurane
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: hematocrit (HCT, equivalent to packed ceⅡ volume), hemoglobin (HGB), erythrocyte count (RBC), total leukocyte (WBC), platelet count (PLAT), the differential Ieukocyte count, and a morphologic examination. Slides of blood smears were prepared and saved for all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the terminal sacrifice after 4 weeks.
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: urea nitrogen (UN), alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), alkaline pbosphatase activity (AP), glucose (GLUC), total protein (TP), albumin (ALB), globulin (GLOB, calculated), total bilirubin (TBILI), phosphorus (PHOS), calcium (CALC), sodium (Na), potassium (K) and chloride (CL)

URINALYSIS: Yes
- Time schedule for collection of urine: during the fourth week of exposure.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: color, character, bilirubin, glucose, ketones, blood, pH, protein, urobilinogen, specific gravity. Microscopic examination was performed on sediments of pooled samples by exposure group.

NEUROBEHAVIOURAL EXAMINATION: performed during clinical examinations
Sacrifice and pathology:
- Animals in the main group were necropsied the day following the last exposure. All animals fasted overnight prior to necropsy
- All animals were weighed, anesthetized with methoxyflurane, and humanely euthanized
- Organ weights were taken from brain, lungs, Iiver, kidneys, adrenals and testes, plus an in situ examination of the eyes by a glass-side technique with fluorescent illumination.
- All animals were examined for gross pathological alterations.
- Histophathological examination of tissues and all identified gross lesions of rats in the 0 and 70 mg/m³ group
- Microscopic examination of respiratory tract tissue from rats exposed to 2 and 12 (nominal) mg/m³ was conducted because exposure-related effects were noted in animals from the highest exposure group.
- Satellite group animals were necropsied 10 weeks after the last exposure according to the pathology procedure described above. Microscopic examination of the respiratory tract from control and 70 mg/m³ exposed rats was conducted.
Other examinations:
N.A.
Statistics:
- Descriptive statistics only (means and standard deviations) are reported for chamber concentration, temperature, relative humidity, airflow, WBC differential counts, and RBC indices
- All parameters examined statistically were first tested for equality of variance using Bartlett's test (if the results from Bartletts test were significant, then the data for the parameter were subjected to a transformation to obtain equality of the variances. When Bartlett's test was satisfied no further transformations were to be applied, or, if none of the transformations resulted in homogeneous variances, the transformed data or raw data with the lowest Bartlett's statistic was used. The selected form ofthe data was then subjected to the appropriate parametric analysis as described below.
- In-life body weights were evaluated using a three-way repeated measures analysis of variance (RM-ANOVA) for time, sex, and dose
- Terminal body weight, organ weight (absolute and relative, excluding ovaries and testes), hematologic parameters (excluding differential WBC and RBC indices), clinical chemistry parameters, and urine specific gravity were evaluated using a two-way ANOVA with the factors of sex and dose. For these parameters the first exmination was whether the sex-dose interaction was significant. If so, an one-way ANOVA was done separately for each sex.
- Comparisons of individual dose groups to the control group were made with Dunett's test only when a statisticaⅡy significant dose effect existed
- Results for ovaries and testes weight (absolute and relative) were analyzed using a one-way ANOVA.If significant dose effects were determined in the one way ANOVA, then separate doses were to be compared to controls using Dunnett's test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
See details below
Mortality:
no mortality observed
Description (incidence):
See details below
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See details below
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See details below
Urinalysis findings:
no effects observed
Description (incidence and severity):
See details below
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See details below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See details below
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details below
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no clinically observed exposure related effects. There were sporadic observations of fur soiling (no single animal was repeatedly soiled). This was considered to be a normal, occasional consequence of restraint in rats acclimated for the nose-only exposure.

BODY WEIGHT AND WEIGHT GAIN
There was a statistically significant difference in the 10 and 70 mg/m³ exposure groups, but no statistically significant difference when the satellite group animals only were compared. The statistical analysis apparently reflects the lower mean body weight of the control males during the 4-week exposure period, and the lower mean body weight of both the control and 2 mg/m³ exposed males after the exposure period. These statistical differences are not likely to be exposure-related and were not considered to be biologically significant in any case.

HAEMATOLOGY
The main effect on hematology was an increase in neutrophil to lymphocyte ratio, reflecting a pulmonary inflammatory response, which was observedalso histologically. The neutrophil:leukocyte ratios increased in an exposure-related manner, except for females in the 2 mg/m³ group after the 10-week post exposure period, there was a numeric decrease relative to controls. Since females held for a 10-week post exposure period tended to have the greatest histopathological response, the numeric changes in the ratio at 2 mg/m³ were not considered biologically significant. A statistical increase in white blood cells (WBC) of rats exposed to 70 mg/m³ group was thought to reflect neutrophilia, that was more accurately depicted by the differential count. There were no apparent effects on leukocyte, erythrocyte, or platelet morphology, although there was increased variability in erythrocyte morphology in all groups of male rats.

CLINICAL CHEMISTRY
There were minor differences in total protein and globulin values that were statistically identified in 0 vs 70 mg/m³ female groups only. These were not a primary significant AlN effect, and may not have been exposure-related at all. The 10 mg/m³ exposed group had significant histopathologic effects, but no changes in these clinical laboratory parameters. There were no other statistical effects on other clinical chemistry values.

URINALYSIS
No effects were seen on any urinalysis parameter examined.

ORGAN WEIGHTS
Organ weight effects were restricted to alterations related to lung weight. Lung weights were increased at 4 weeks in the 70 mg/m³ (both sexes) and in the 10 mg/m³ exposed females. Relative lung weight means were increased in the 70 mg/m³ exposure groups.
Ten weeks after exposure, there was a significant increase in mean lung weights of all exposed rats. Lung weight relative to body weight increased in females (2 mg/m³ and greater) and males (10 mg/m³ and greater). There was a more pronounced effect on lung weights after 10 weeks and a slight effect in 2 mg/m³ exposed rats. Females may be more sensitive than males.

PATHOLOGY
Main Group:
After 4 weeks, effects were seen in the lungs, mediastinal lymph nodes (see Table 1 & 2) and nasal tissues. Brown granular particulate material was seen in lung and lymphoid tissues. In the lungs of rats exposed to 70 mg/m³, approximately 25% of the test material was free-either unphagocytized or freed from dead macrophages- and there was a slight subacute inflammatory response in 80% of the lung. This response was characterized as proliferative pneumonitis because of the proliferation of alveolar type II cells lining affected alveoli. Rats exposed to 2 and 10 mg/m³ showed little or no evidence of inflammation.
At necropsy, the mediastinal lymph nodes were enlarged and pale in 4/5 high exposed females. This change correlated histologically with reactive hyperplasia and the presence of test material within aggregates of macrophages in the nodes of 70 mg/m³ of rats of both sexes.
In nasal tissues there was a slight goblet cell hyperplasia noted in all high dose rats. This was considered to be non-specific and of little toxicologic significance.
High dose male rats exhibited slight chronic inflammation of the larynx, versus one slight chronic inflammation in male controls.Inflammation was associated with focal areas of mineralization. These foci of mineralization are very common spontaneous findings in Fischer-344 rats. Slight epithelial hyperplasia was observed in 4/5 high dose males and none at lower doses. Laryngeal lesions was only increased coincident with exposure levels in males.

Ten-Week Post-Exposure Group:
Test material was not appreciably cleared and inflammatory reaction in the lungs and mediastinal lymph nodes intensified (see Table 1 & 2). Grossly, the lungs of high dose rats of both sexes had an accentuated lobular pattern. Microscopically, the pattern was defined by normal alveoli clustered nearer the bronchiole, while the more peripheral portions of acinar unit tended to be filled with proteinaceous material, cellular debris, macrophages, and a small number of neutrophils, and test material. Alveolar septal thickening was marked due to prominent alveolar type II cell proliferation and interstitial congestion and thickening. Reactive hyperplasia of bronchial associated lymphoid tissue was quite marked, with numerous aggregates of macrophages containing test material scattered within the tissue. Perivascular lymphoid tissue was moderately increased.
After 10 weeks, most of the test material was extracellular. 90% of the high dose group lung parenchyma was severely affected.
The 2 and 10 mg/m³ groups had similar effects, but to much smaller degrees. There was one important qualitative difference from the high exposure group: the test material was almost all within alveolar macrophages rather than free in alveolar spaces, although at 10 mg/m³ some macrophages laden with the test material were disintegrating. Tissue response was less intense at lower exposures. Pathological changes in mediastinal lymph nodes extended to lower exposure groups. Gross enlargement of nodes was observed in all 70 mg/m³ exposed rats and most 10 mg/m³ exposed rats. Gross lesions correlated histologicaⅡy with reactive hyperplasia and the presence of test material in aggregates of macrophages in all 70 and 10 mg/m³ rats, and one 2 mg/m³ female rat.
Nasal tissue changes resolved substantially. Slight goblet cell hyperplasia was seen only in two high exposed females. Focal very slight acute inflammation was seen in one high dose male. Laryngeal lesions were similar to that observed in the main group, but without correlation to exposure level or to sex.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
(local)
Effect level:
2 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on mild adverse effects in the lungs, but alveolar macrophages had engulfed aⅡ the foreign material, which is an important step in the clearance process.
Dose descriptor:
LOAEC
Remarks:
(local)
Effect level:
10 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Remarks:
(systemic)
Effect level:
70 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lack of adverse effects (besides effects in the lung) at the highest concentration tested.

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/m³ air
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes

Any other information on results incl. tables

Table 1: Significant Lung Histopathology
SEX M M M M F F F F
AlN Conc. (mg/m³) 0 2 10 70 0 2 10 70
Number examined 5 5 5 5 5 5 5 5
       
Lungs, 4-week main study    
Pneumonitis - proliferative slight 0 0 0 5 0 0 0 5
Macrophages with Foreign Material (MFM) very slight 0 5 0 0 0 5 0 0
slight 0 0 5 0 0 0 5 0
Macrophages with Foreign Material and Free Foreign Material (MFM/FFM) Severe 0 0 0 5 0 0 0 5
 
Lungs, 10-Week Post-Exposure    
Pneumonitis - proliferative very slight 0 4 0 0 0 5 0 0
slight 0 1 0 0 0 0 0 0
moderate 0 0 5 0 0 0 5 0
severe 0 0 0 5 0 0 0 5
MFM very slight 0 5 0 0 0 5 0 0
MFM/FFM slight 0 0 5 0 0 0 5 0
        severe 0 0 0 5 0 0 0 5

Table 2: Mediastinal Lymph Node (L.N.) Significant Findings
SEX M M M M F F F F
AlN Conc. (mg/m³) 0 2 10 70 0 2 10 70
Number examined 5 5 5 5 5 5 5 5
       
Mediastinal L.N., 4-week main study    
Hyperplasia -reactive 0 0 0 5 0 0 0 4
Macrophages with Foreign Material (MFM) very slight 0 0 1 0 0 0 0 0
slight 0 0 0 0 0 0 0 1
moderate 0 0 0 5 0 0 0 4
 
Mediastinal L.N., 10-Week Post-Exposure    
Hyperplasia -reactive 0 0 5 5 0 1 5 5
MFM slight 0 1 0 0 0 1 0 0
moderate 0 0 5 0 0 0 5 0
severe 0 0 0 5 0 0 0 5

Applicant's summary and conclusion

Conclusions:
Aluminum nitride elicits local effects in the lung at concentrations saturating the lung clearance in rats. No adverse systemic effects have been reported at the highest attained concentration of 70 mg/m³.
Executive summary:

In a subacute inhalation toxicity study, aluminium nitride (AlN) was administered to 10 Fischer 344 rats/sex/concentration by nose only exposure at concentrations of 0, 2, 10, and 70 mg/m³ for 6 hours/day for 5 days/week for a total of 19 exposures during a 4 week study. The average Mass Median Aerodynamic Diameter (MMAD) was 1.5 μm; 84% of the aerosol mass consisted of particles less than 2.4 μm. Five rats/sex/exposure concentration were evaluated by pathology immediately after the 4 week exposure period, and five more rats/sex/exposure concentration were held for approximately 10 weeks after the last exposure, and were then evaluated by pathology. In addition to gross and histopathologic evaluations, there were clinical observations and determinations of body weights, clinical chemistry, hematology, urinalysis, and organ weights. Because the only exposure-related effect were related to the respiratory system in rats evaluated at 4 weeks, evaluations were focused on rats that were held for 10 week post-exposure.

After four weeks, there was a mild inflammatory response in the lungs of rats exposed to 70 mg/m³. In the 2 and 10 mg/m³ groups, there was little or no evidence of a tissue reaction in the lungs, other than phagocytosis. At the lower exposure concentrations, virtually all of the visible foreign material in the lung parenchyma was contained within alveolar macrophages. This foreign material was presumably AlN or an AlN degradation product. At 70 mg/m³, approximately 25% of the foreign material was "free” either phagocytized or freed from dead macrophages.

Following the 10 week post-exposure period, histologic examination of lungs from rats in the 2 mg/m³ group indicated that foreign material was present within the alveolar macrophages, and there were widely scattered small areas of mild inflammation characterized by slight alveolar type II cell proliferation and occasional neutrophils. At 10 mg/m³, the changes described above were increased, and there were reactive changes in the bronchial associated lymphoid tissue (BALT). At 70 mg/m³, much of the foreign material was trapped within proteinaceous material filling many of the alveolar spaces.

Alveolar type II cell proliferation and the BALT reaction were marked. Lung weights and blood neutrophil:leukocyte ratios were increased in most groups with pulmonary inflammation. Overall the reaction was characterized as a proliferative pneumonitis.

In rats exposed to 70 mg/m³ and evaluated immediately after 4 weeks of exposure, there were signs of upper respiratory tract irritation which included slight goblet cell hyperplasia of the nasal turbinates. Effects on the upper respiratory tract were largely resolved at 10 weeks post-exposure, and there were no upper respiratory tract effects in rats exposed to 2 or 10 mg/m³.

Proliferative pneumonitis was more severe after the 10 week post-exposure period. Excessive exposure to AIN exceeded physiologic lung clearance mechanisms, as suggested by the free foreign material in pulmonary alveoli of rats exposed to this concentrations of AIN aerosol. This may in part account for the more pronounced effects at 10 weeks post-exposure.

Overall, AlN predominantly affected the alveolar macrophages. The NOAEC for male and female rats was 2 mg/m³ based on mild adverse effects in the lungs and observed alveolar macrophages engulfing foreign material, which is an important step in the clearing process. The adverse effects at higher concentrations are considered to justify CLP classification for specific target organ toxicity (STOT RE2, lung, local).

This sub-acute toxicity study in the rat is acceptable and satisfies the guideline requirement accordign to OECD 412 for a sub-acute inhalation study in the rat.