Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Pulmonary reaction to metallic aluminium powders.
Author:
Gross P, Harley R, de Treville RTP
Year:
1973
Bibliographic source:
Arch Environ Health.26:227-236.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
: Only one sex of animals; Number of animals per group (sex/dose/timepoint); Outcomes assessed (lack of observations of body weight and other clinical signs); lack of information on animal husbandry
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Aluminium powder (pyro)
Supplier: from Britain
Purity: not reported (NR)
- 16.6 % Al2O3
- 0.18% “total grease”
Batch number: NR
Storage: NR

Name: Aluminium powder (atomized)
Supplier: not specified, from the “American continent”.
Purity: NR
- 1.1% Al2O3
- 0% “total grease”
Batch number: NR
Storage: NR

Name: Aluminium powder (flake)
Supplier: not specified, from the “American continent”.
Purity: NR, %
- Al2O3 unclear
- “total grease” unclear
Batch number: NR
Storage: NR

Name: Aluminium oxide dust
Supplier: NR
Purity: 99.8% pure
Batch number: NR
Storage: NR

Test animals

Species:
rat
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
Details on test animals and environmental conditions: no data
It is unclear whether the animals remained in the inhalation chambers even when the exposure was not occurring.

Diet and water: NR

Acclimation and monitoring animal health:
No information was provided on acclimation or animal care.

Administration / exposure

Route of administration:
other: Inhalation: dust and Intratracheal injections
Type of inhalation exposure:
whole body
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: No information was provided on the MMAD and GSD.

Further detail on particle characteristics (e.g. shape):
Particle size by count was provided for the size ranges < 1.0 µm; 1 to 4 µm; and > 4 µm.
(1) British pyro powder
Shape: flake-like
Size: < 1 µm 4.2%, 1 – 4 µm 87.3%, > 4 µm 8.5%
Mean diameter: 2.49 µm
Specific surface area: 10.4 m²/g

(2) US –source atomised particles.
Shape: “spherical”
Size: < 1 µm 1.5%, 1 – 4 µm 95.6%, > 4 µm 2.9%;
Mean diameter: 2.22 µm
Specific surface area: 0.8 m²/g

(3) US-source flake powder
Shape: flake
Size: < 1 µm 0.0%, 1 – 4 µm 28.6%, > 4 µm 71.4%;
Mean diameter: 4.85 µm
Specific surface area: 8.4 m²/g

(4) Negative control: aluminium oxide dust.
Shape: not stated
Size: < 1 µm 66%, 1 – 4 µm 25%, > 4 µm 9%;
Mean diameter: 0.80 µm
Specific surface area: 6.3 m²/g
Details on inhalation exposure:
Further details on inhalation exposure:
The chambers were approximately 1.2 m³ in volume. Moisture was removed using anhydrous calcium chloride. Powders were dispersed through the chambers by means of a dust-feed mechanism (Wright). Air flow was limited to 10 litres/min to attain high dust concentrations.

Details on Intratracheal Instillation:
A suspension of the dust in tap water was instilled intratracheally. Concentrations were used such that 1 mL of the suspension contained the required dose. Injections were performed under anaesthetic (ether) using an illuminated laryngeal speculum to facilitate the introduction of the 18-gauge, blunt needle.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The air flow through the chambers was monitored with outflowing chamber air forced through 20.3 x 25.4 cm filter (Millipore) filters before ventilation. The filters were used to gravimetrically estimate the average dust concentration in the chamber each day. The data were not reported however.
Duration of treatment / exposure:
Inhalation
Rats in the 50 and 100 mg/m³ chambers were exposed for 6 months. Exposure duration was 12 months for the animals at the lower aluminium powder concentrations of 15 and 30 mg/m³.

The aluminium oxide control rats were exposed to 75 mg/m³ for six months. An additional 30 rats and 12 guinea pigs were exposed to 30 mg/m³ of aluminium oxide dust for a year.
Frequency of treatment:
Inhalation
6 hr/day; 5 days a week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Inhalation
Basis:
other: Pyro powder: Rats: 15, 30, 50 and 100 mg/m³
Remarks:
Doses / Concentrations:
Inhalation
Basis:
other: Atomized powder: Rats: 15, 30, 50 and 100 mg/m³
Remarks:
Doses / Concentrations:
Inhalation
Basis:
other: Flaked powder: Rats: 15 and 30 mg/m³
Remarks:
Doses / Concentrations:
Inhalation
Basis:
other: Aluminium oxide controls: Rats: 30 and 70 mg/m³
Remarks:
Doses / Concentrations:
Intratracheal instillation
Basis:
other: Pyro powder: Rats: 2, 4, 5, 6, 12, 24, 100 mg; Pyro powder (degreased): Rats: 100 mg; Atomized powder: Rats: 2, 4, 5, 6, 12, 24, 100 mg; Flaked powder: Rats: 2, 4, 5, 6, 12, 24 mg; Plus water controls: 0 mg
No. of animals per sex per dose:
Inhalation
Rats:
30 rats were exposed to pyro powder at each 15, 30, 50 and 100 mg/m³
30 rats were exposed to atomized metal powder at each 15, 30, 50 and 100 mg/m³
30 rats were exposed to flake powder at 15 and 30 mg/m³
30 rats were exposed to aluminium oxide dust at 30 and 70 mg/m³

- 5 animals were sacrificed per time point (6, 8, 12 and 18 months).

Intratracheal instillation **
15 rats were allocated to each dose for the pyro, atomized and flaked powders. With the exception of the highest dose level, 1 to 5 animals were sacrificed at 6 months and 7 to 10 animals at 12 months.

At the 100 mg/m³ dose level for the pyro powder, 15 animals were dosed, 4 were sacrificed at 2 months, 4 at 4 months and 7 at 6 months.

At the 100 mg/m³ dose level for the atomized powder, 15 animals were dosed, 3 were sacrificed at 2 months, 3 at 4 months and 2 at 6 months.
Control animals:
yes
Details on study design:
Control animals:
50 rats were untreated (laboratory controls). Five animals were examined per time point.
Aluminium oxide (numbers and dosing described above) were included as “non-fibrogenic” controls.
For the intratracheal instillation group, 15 rats were included as vehicle controls.

No information was provided on the method used to allocate the animals to groups.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
Observations and examinations performed:
No information was provided on observations to monitor animal health but mortality was recorded.

Frequency of the observations and examinations:
For the inhalation exposure, pathological examinations took place at 6, 8, 12 and 18 months into the experiment for the 50 and 100 mg/m³ aluminium powder dose groups and the 70 mg/m³ aluminium oxide dose group (i.e. 0, 2, 6 and 12 months after cessation of exposure). Kills of the lower dose animals took place at 6 and 12 months (0 and 6 months post-exposure).
For the intratracheal instillation, see ** above.
Sacrifice and pathology:
The method used to sacrifice the animals was not reported in the article.
Histopathological examinations of lung tissue were conducted using sections cut in triplicate and embedded in paraffin blocks. One section was stained with eosin to show aluminium particles, a second section was stained with hematoxylin-eosin, and a third section with PAS or van Gieson. To show cellular components and stromal support structures, the hematoxylin-eosin stained sections were photographed then decolorized and impregnated with silver (Gordon and Sweets method) before another photograph was taken. Aluminium particles were removed prior to this procedure using 10% sodium bisulfite.
Other examinations:
No data.
Statistics:
No information was provided on statistical methods used for comparing mortality rates.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
not specified
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:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
A higher rate than ideal was observed. As high rates were observed in control groups as well as treated groups and appeared to show no relationship to dose, deaths were unlikely to be simply related to the dust exposure. The authors suggest an effect of crowding and low air flow in the chambers. Quantitatively, the mortality results from this study are not reliable. The absence of observations of clinical signs also suggests that caution ought to be used in interpreting the results.


ORGAN WEIGHTS
Lung weights were measured but not reported.


HISTOPATHOLOGY: NON-NEOPLASTIC
ADEQUATE
- alveolar proteinosis was observed after 6 months exposure to 15 mg/m³ in rats.
- foci of fibrosis found for pyro Al powder at 50 mg/m³ exposed for 108 days; killed 6 months later.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Pulmonary lymphoid tumors, reticulum cell and lymphosarcoma were noted in both the experimental and control groups. These were interpreted as spontaneous tumors in aging rats not associated with pulmonary dust exposure.




Effect levels

Dose descriptor:
NOAEC
Remarks:
Al2O3 dust
Effect level:
70 mg/m³ air
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: highest dose tested for Al2O3 dust

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Inhalation series:

Mortality: 

Spontaneous deaths were more numerous among all 3 species than ideal. The % of the animals dead at 6 months and 12 months are provided in the table below. The numbers are extracted from Tables 3, 4 and 5 of the publication.

 

Animal

Dust Type

Dose (mg/m³)

Exposure duration

% dead: 6 mos.

% dead: 12 mos.

Rats

Atomised Al

100

6 mos

0

0

 

Atomised Al

50

6 mos

7

25

 

Atomised Al

30

12 mos

0

28

 

Atomised Al

15

12 mos

0

8

 

Pyro Al

100

6 mos

0

40

 

Pyro Al

50

6 mos

0

20

 

Pyro Al

30

12 mos

0

20

 

Pyro Al

15

12 mos

3

36

 

Flake Al

30

12 mos

0

24

 

Flake Al

15

12 mos

0

32

 

Al2O3

70

6 mos

0

0

 

Al2O3

30

12 mos

0

20

Air control

0

6 mos

0

0

Air control

0

12 mos

0

0

 

 

Lung histology

Al-powders:

All three species developed alveolar proteinosis (AP);

 

Rats:

50 and 100 mg/m³ exposed for 6 mths:

Marked AP; but alveolar walls were generally thin and appeared normal;

AP underwent spontaneous resolution with little evidence remaining 1.5 years post-exposure.

15 and 30 mg/m³ for 12 mths:

Moderate AP from 6 to 12 months followed by gradual clearing. Some AP still present at 24 mths.

 

Persistent changes:

Small scattered foci of endogenous lipid pneumonitis (granulomatous inflammation) associated with cholesterol crystals that were not surrounded by AP material. These occurred generally not in regions with dust particles. The foci left collagenous scars.

No carcinoma was observed. Lymphoid tumors, reticulum cell and lymphosarcoma noted in both the treated and control groups. Considered spontaneous by authors and numbers were not provided.

 

 

Al2O3:

Rats:

Small foci concentrated in respiratory bronchioles and alveolar ducts – consisting of clustered alveoli with swollen macrophages engorged with particles; no thickening of alveolar walls evident; no evidence of AP or pnuemonitis.

 

Distribution and clearance of dust:

Dust remained finely dispersed even within the cytoplasm of macrophages.

Rats:

50 and 100 mg/m³ exposed for 6 mths: Clearance by 1.5 years post-exposure

15 and 30 mg/m³ exposed for 12 mths: some finely dispersed Al-powder particles were still evident 1 year post-exposure.

 

There was no dose response evident or noticeable differences in response to the different aluminium powders.

 

The laboratory and the intratracheal injection control did not show evidence of proteinosis.

 

Intratracheal Instillation:

Lung histology

Rats:

Pyro and atomized powder - 100 mg/m³

6 mths: numerous large foci of collagenous fibrosis “sharply circumscribed but highly irregular in outline”; some coalesced; no remaining alveolar structure; coarse bundles of collagen; moderate number of plump connective cells; black pigment masses in connective tissue; alveolar tissue between fibrotic foci usually normal.

12mths: collagenous foci with more fibres and fewer connective cells; similar between the different powders; inter-animal variability in response was evident.

Pyro and atomized powder – 12 to ≤24 mg/m³

Smaller, more widely separate foci that were highly cellular with only a few collagen fibres; foci were concentrated around the respiratory bronchioles and alveolar ducts.

Pyro and atomized powder – ≤12 mg/m³

No significant collagenisation of foci at 6 or 12 mths.

 

Applicant's summary and conclusion

Conclusions:
Intratracheal injection of aluminium powder caused nodular pulmonary fibrosis in the lungs of the rats only at the highest dose administered (100 mg). A fibrotic response was not observed in hamsters indicating inter-species differences in response. 12 mg of dust administered intratracheally did not lead to collagen production in rats or hamsters. The response of hamster and guinea pigs lungs differed from rats. At higher concentrations, hamster and guinea pig lungs developed metaplastic foci of alveolar epithelium that persisted beyond the resolution of alveolar proteinosis and clearance of the dust particles. There was no dose response evident or a noticeable difference between responses to the different aluminium powders.

Progressive fibrosis was not observed in rats on inhalation exposure to the powders indicating that the intratracheal instillation mode of test compound delivery may lead to artifacts not representative of actual inhalation exposures. All three species developed widespread alveolar proteinosis, rats exhibiting the most severe response. The proteinosis resolved progressively after cessation of exposure. The group of rats exposed for 12 months to 15 mg/m³ of aluminium powder showed moderate alveolar proteinosis after only 6 months of exposure.
Executive summary:

Gross et al. (1973) exposed rats, guinea pigs and hamsters to three different aluminium powders (British pyro powder, a US-flake powder, and a US-source atomized powder with approximately spherical particles) and also aluminium oxide dust, included as a negative “non-fibrogenic control”. The Al2O3content was 16.6% for the British pyro powder, not stated for theflake powder and 2.9% for the atomized powder. The doses administered by inhalation ranged from 15 to 100 mg/m³, 6 hours per day, 5 days per week for either 6 or 12 months. Thirty rats were exposed to pyro powder at each 15, 30, 50 and 100 mg/m³, 30 rats were exposed to atomized metal powder at each 15, 30, 50 and 100 mg/m³, 30 rats were exposed to flake powder at 15 and 30 mg/m³, and 30 rats were exposed to aluminium oxide dust at 30 and 70 mg/m³. Five rats were sacrificed per time point (6, 8, 12 and 18 months). Thirty hamsters were exposed to pyro powder at 50 and 100 mg/m³, 30 hamsters were exposed to atomized powder at 50 and 100 mg/m³, and 30 hamsters were exposed to aluminium oxide at 70 mg/m³. Between 15 and 25 guinea pigs were exposed to each of the aluminium powders at 15 and 30 mg/m³. Twelve guinea pigs were exposed to aluminium oxide dust at 30 mg/m³. The chambers were approximately 1.2 m³ in volume, moisture was removed using anhydrous calcium chloride andpowders were dispersed through the chambers by means of a dust-feed mechanism (Wright). Air flow was limited to10 litres/min to attain high dust concentrations. 

 

The dusts, suspended in tap water, were also administered by intratracheal instillation to different groups of animals. Concentrations were used such that 1mL of the suspension contained the required dose. Injections were performed under anaesthetic (ether) using an illuminated laryngeal speculum to facilitate the introduction of the 18-gauge, blunt needle. A tap water “vehicle” control group was included. For intratracheal instillation, 15 rats and 15 hamsters were allocated to each dose for the pyro, atomized and flaked powders. With the exception of the highest dose level, 1 to 5 animals were sacrificed at 6 months and 7 to 10 animals at 12 months post-exposure. At the 100mg/m³ dose level for the pyro powder, 15 animals were dosed, 4 were sacrificed at 2 months, 4 at 4 months and 7 at 6 months. At the 100mg/m³ dose level for the atomized powder, 15 animals were dosed, 3 animals were sacrificed at 2 months, 3 animals at 4 months and 2 animals at 6 months.

 

Mortality was reported but no data on clinical signs, body weight, or organ weights was provided. Histopathological examinations of the lungs were conducted on sections cut in triplicate from lung tissue stained with either eosin alone to show aluminium particles, hematoxylin-eosin,or PAS/ van Gieson. To show cellular components and stromal support structures, the hematoxylin-eosin stained sections were examined before and after decolorization and impregnation with silver (Gordon and Sweets method).

 

Intratracheal injection of the aluminium powders caused nodular pulmonary fibrosis in the lungs of the rats only at the highest dose administered (100mg). A fibrotic response was not observed in hamsters indicating inter-species differences in response. 12mg of dust administered intratracheally did not lead to collagen production in rats or hamsters. The response of hamster and guinea pigs lungs differed from rats. At higher concentrations, hamster and guinea pig lungs developed metaplastic foci of alveolar epithelium that persisted beyond the resolution of alveolar proteinosis and clearance of the dust particles. 

 

Progressive fibrosis was not observed in rats on inhalation exposure to the powders indicating that the intratracheal instillation mode of test compound delivery may lead to artifacts not representative of physiologically relevant exposures. There was no dose response evident or a noticeable difference between responses to the different aluminium powders. All three species developed widespread alveolar proteinosis, rats exhibiting the most severe response. However, alveolar walls appeared thin and normal. The proteinosis resolved progressively after cessation of exposure. Small scattered foci of endogenous lipid pneumonitis (granulomatous inflammation) developed associated with cholesterol crystals that were not surrounded by alveolar proteinaceous material. These effects generally occurred in regions not associated with dust particles and left small collagenous scars. The group of rats exposed for 12 months to 15mg/m³ of aluminium powdershowed moderate alveolar proteinosis after 6 months of exposure. Granulomatous inflammation was observed at 50 mg/m³ after about 3 months of exposure.

 

Overall, there was no consistent relationship between dose and severity of response for any of the aluminium powders. The results showed no clear difference in reaction to the different powders. The results from this study do not provide evidence to support a progressive fibrotic response on inhalation exposure to aluminium powder. No alveolar proteinosis or thickening of alveolar walls was observed in rats, hamsters or guinea pigs exposed to Al2O3dust (66% <1μm) included in the study as a “non-fibrogenic” control. 

 

The reason for the high and variable rates of mortality in this study is unclear and is a limitation of the study. Several endpoints specified in the 90-day inhalation toxicity guideline (OECD TG 413) were not assessed, particularly body and organ weights. The study design and animal husbandry were not described in sufficient detail. Considering reliability for use in the hazard identification, a Klimisch Score of 2 is appropriate for the lung pathology results and a Score of 3 for the mortality results.