Aluminium oxide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

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

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

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

Species:

hamster

Strain:

not specified

Sex:

not specified

Details on test animals or test system 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: no data

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

Route of administration:

other: Inhalation: dust and Intratracheal injections

Type of inhalation exposure:

whole body

Vehicle:

not specified

Remarks:

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:

6 months

Frequency of treatment:

Inhalation
6 hr/day; 5 days a week

Dose / conc.:

50 mg/m³ air

Remarks:

pyro powder, atomized powder

Dose / conc.:

100 mg/m³ air

Remarks:

pyro powder, atomized powder

Dose / conc.:

75 mg/m³ air

Remarks:

aluminium oxide

No. of animals per sex per dose:

Inhalation
Hamsters:
30 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
30 hamsters were exposed to aluminium oxide at 70 mg/m³

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.

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:
25 hamsters 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, 5 hamsters were included as vehicle controls.

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

Positive control:

No.

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 75 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.

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

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

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
- foci of fibrosis found for pyro Al powder at 50 mg/m³ exposed for 108 days; killed 6 months later.
- alveolar proteinosis was observed after 2 to 3 months of exposure in two guinea pigs.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
The authors reported no tumors in the lungs of hamsters in the inhalation series.

At intratracheal instillation doses ≤ 24 mg of aluminium powder,
“striking metaplasia of alveolar epithelium” (p231 of article) was observed in the lungs of hamsters. “Cuboidal metaplasia” of the alveolar epithelium was observed in hamsters exposed to the highest concentrations of aluminium powder (Fig. 6 and Fig. 7 of the article). There is mention of these effects only in treated animals.

Dose descriptor:

LOAEC

Remarks:

local effects

Effect level:

100 mg/m³ air

Based on:

test mat.

Remarks:

Al powder

Sex:

not specified

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

>= 75 mg/m³ air

Based on:

test mat.

Remarks:

Al2O3 dust

Sex:

not specified

Basis for effect level:

other: no adverse effect observed at the highest dose tested

Critical effects observed:

not specified

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/m^3)	Exposure duration	% dead: 6 mos.	% dead: 12 mos.
Hamsters	Atomised Al	100	6 mon	0	7
	Atomised Al	50	6 mon	0	31
	Pyro Al	100	6 mon	20	60
	Pyro Al	50	6 mon	7	25
	Al2O3	75	6 mon	6	18
	Air control	0	6 mon	4	13

 

 

Lung histology

Al-powders:

Hamsters developed alveolar proteinosis (AP);

  

Hamsters:

50 and 100 mg/m³ for 6 mths:

Mild AP at the end of exposure. No AP or Al-powder evident 3 mths post-exposure.

Persistent changes: foci of metaplasia of alveolar epithelium – gland-like structures in alveoli opening off respiratory bronchioles and alveolar ducts. These effects are described for treated animals only.

No tumors were observed in the hamster lungs.

 

 

 

Intratracheal Instillation:

Lung histology

 

Hamsters:

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

Smaller, more widely separate foci that were highly cellular with no collagen fibres; no evidence of inflammatory response; foci were concentrated around the respiratory bronchioles and alveolar ducts; presence of striking metaplasia of the alveolar epithelium giving the tissue a multiglandular appearance.

Pyro and atomized powder – ≤12 mg/m³

No significant collagenisation of foci at 6 or 12 mths.

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 75 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 75 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 and powders 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 15 mg/m³ of aluminium powder showed 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 chronic toxicity guideline (OECD TG 452) 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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

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

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Details on test animals or test system 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: no data

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

Route of administration:

other: Inhalation: dust and Intratracheal injections

Type of inhalation exposure:

whole body

Vehicle:

not specified

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.

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:

12 months

Frequency of treatment:

Inhalation
6 hr/day; 5 days a week

Dose / conc.:

15 mg/m³ air

Remarks:

pyro powder, atomized powder, flake powder

Dose / conc.:

30 mg/m³ air

Remarks:

pyro powder, atomized powder, flake powder

Dose / conc.:

30 mg/m³ air

Remarks:

aluminium oxide

No. of animals per sex per dose:

Inhalation
Guinea pigs:
14 and 26 guinea pigs were exposed to pyro powder at 15 and 30 mg/m³, respectively.
15 and 19 guinea pigs were exposed to atomized powder at 15 and 30 mg/m³, respectively.
21 and 25 guinea pigs were exposed to flake powder at 15 and 30 mg/m³, respectively.
12 guinea pigs were exposed to aluminium oxide at 30 mg/m³.

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.

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 100mg/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:
12 guinea pigs were untreated (laboratory controls). Five animals were examined per time point.
Aluminium oxide (numbers and dosing described above) were included as “non-fibrogenic” controls.

Positive control:

No.

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 and 12 months (0 and 6 months post-exposure).

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.

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

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

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 2 to 3 months of exposure in two guinea pigs.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
The authors reported no tumors in the lungs of guinea pigs in the inhalation series.

“Cuboidal metaplasia” of the alveolar epithelium was observed in guinea pigs exposed to the highest concentrations of aluminium powder (Fig. 6 and Fig. 7 of the article). There is mention of these effects only in treated animals.

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

>= 30 mg/m³ air

Based on:

test mat.

Remarks:

Al2O3 dust

Sex:

not specified

Basis for effect level:

other: no adverse effect observed at the highest dose tested

Critical effects observed:

not specified

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.
Guinea pigs	Atomised Al	30	12 mos	42	47
	Atomised Al	15	12 mos	33	53
	Pyro Al	30	12 mos	69	81
	Pyro Al	15	12 mos	14	50
	Flake Al	30	12 mos	92	96
	Flake Al	15	12 mos	62	71
	Al2O3	30	12 mos	8	17
	Air control	0	12 mos	23	23

 

 

Lung histology

Al-powders:

Guinea pigs developed alveolar proteinosis (AP);

 

Guinea pigs:

AP that developed was not severe and cleared readily on cessation of exposure. Dust particles were also readily cleared.

Persistent changes:

similar to hamsters but altered alveoli contained dust-filled macrophages to a greater extent than hamsters. These effects are observed for treated animals only.

No tumors were observed in the guinea pig lungs.

 

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 Al2O3 content was 16.6% for the British pyro powder, not stated for the flake 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 75 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 75 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 and 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. 

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 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 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 (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 (i.e. 100 mg/m³ for hamsters, unclear for guinea pigs), 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 15 mg/m³ of aluminium powder showed 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 Al2O3 dust (66% <1 μm) 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 chronic toxicity guideline (OECD TG 452) 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.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity, other

Remarks:

dust and Intratracheal injections

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1973

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD TG 452

Deviations:

yes

Remarks:

: number of animals to low (30 instead of 50 per sex and dose); duration only 1 year instead of 2 years.

GLP compliance:

not specified

Species:

rat

Strain:

not specified

Sex:

not specified

Details on test animals or test system 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.

Route of administration:

other: inhalation: dust and Intratracheal injections

Type of inhalation exposure (if applicable):

whole body

Vehicle:

not specified

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 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 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.

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
According to Table 2 of the article -
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 were exposed to 30 mg/m³ of aluminium oxide dust for a year.

Frequency of treatment:

Inhalation:
6 hr/day; 5 days a week

Dose / conc.:

15 mg/m³ air

Remarks:

pyro powder, atomized powder, flake powder

Dose / conc.:

30 mg/m³ air

Remarks:

pyro powder, atomized powder, flake powder

Dose / conc.:

50 mg/m³ air

Remarks:

pyro powder, atomized powder

Dose / conc.:

100 mg/m³ air

Remarks:

pyro powder, atomized powder

Dose / conc.:

30 mg/m³ air

Remarks:

aluminium oxide

Dose / conc.:

75 mg/m³ air

Remarks:

aluminium oxide

No. of animals per sex per dose:

Inhalation:
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 75 mg/m³

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 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 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.

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).

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 data.

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not specified

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

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

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.


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.

Dose descriptor:

LOAEC

Remarks:

local effects

Effect level:

15 mg/m³ air

Based on:

test mat.

Remarks:

Al powder

Sex:

not specified

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

>= 75 mg/m³ air

Based on:

test mat.

Remarks:

Al2O3 dust

Sex:

not specified

Basis for effect level:

other: no adverse effect observed at the highest dose tested

Critical effects observed:

not specified

Mortality: 

Animal	Dust Type	Dose (mg/m^3)	Exposure duration	% dead: 6 mos.	% dead: 12 mos.
Rat	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	75	6 mos	0	0
	Al2O3	30	12 mos	0	20
	Air control	0	6 mos	0	0
	Air control	0	12 mos	0	0

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 interspecies 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 Al2O3 content was 16.6% for the British pyro powder, not stated for the flake 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 75 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 75 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 and 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. 

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 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 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 (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 (i.e. 100 mg/m³ for hamsters, unclear for guinea pigs), 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 15 mg/m³ of aluminium powder showed 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 Al2O3 dust (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 chronic toxicity guideline (OECD TG 452) 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.

Cross-reference 4

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity, other

Remarks:

inhalation: dust and Intratracheal injections

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1973

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD TG 452

Deviations:

yes

Remarks:

: number of animals to low (30 instead of 50 per sex and dose); duration only 1 year instead of 2 years.

GLP compliance:

not specified

Species:

hamster

Strain:

not specified

Sex:

not specified

Details on test animals or test system 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.

Route of administration:

other: inhalation: dust and Intratracheal injections

Type of inhalation exposure (if applicable):

whole body

Vehicle:

not specified

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 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 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.

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
According to Table 2 of the article -
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 were exposed to 30 mg/m³ of aluminium oxide dust for a year.

Frequency of treatment:

Inhalation:
6 hr/day; 5 days a week

Dose / conc.:

50 mg/m³ air

Remarks:

pyro powder, atomized powder

Dose / conc.:

100 mg/m³ air

Remarks:

pyro powder, atomized powder

Dose / conc.:

75 mg/m³ air

Remarks:

aluminium oxide

No. of animals per sex per dose:

Inhalation:
30 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
30 hamsters were exposed to aluminium oxide at 75 mg/m³

Intratracheal instillation:
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.

Control animals:

yes

Details on study design:

Control animals:
25 hamsters 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, 5 hamsters were included as vehicle controls.

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

Positive control:

No.

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).

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.

Statistics:

No data.

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not specified

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

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

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
- foci of fibrosis found for pyro Al powder at 50 mg/m³ exposed for 108 days; killed 6 months later.
- alveolar proteinosis was observed after 2 to 3 months of exposure in two guinea pigs.

LUNG HISTOLOGY
Inhalational series:
Al-powders:
Alveolar proteinosis (AP) was found.

50 and 100 mg/m³ for 6 mths:
Mild AP at the end of exposure. No AP or Al-powder evident 3 mths post-exposure.
Persistent changes: foci of metaplasia of alveolar epithelium – gland-like structures in alveoli opening off respiratory bronchioles and alveolar ducts. These effects are described for treated animals only at the “higher concentration”.
No tumors were observed in the hamster lungs.

Intratracheal Instillation:
Lung histology
Pyro and atomized powder - 12 to ≤ 24mg/m³
Smaller, more widely separate foci that were highly cellular with no collagen fibres; no evidence of inflammatory response; foci were concentrated around the respiratory bronchioles and alveolar ducts; presence of striking metaplasia of the alveolar epithelium giving the tissue a multiglandular appearance.
Pyro and atomized powder – ≤ 12mg/m³
No significant collagenisation of foci at 6 or 12 mths.

Dose descriptor:

LOAEC

Remarks:

local effects

Effect level:

100 mg/m³ air

Based on:

test mat.

Remarks:

Al powder

Sex:

not specified

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

>= 75 mg/m³ air

Based on:

test mat.

Remarks:

Al2O3 dust

Sex:

not specified

Basis for effect level:

other: no adverse effect observed at the highest dose tested

Critical effects observed:

not specified

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 interspecies 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 Al2O3 content was 16.6% for the British pyro powder, not stated for the flake 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 75 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 75 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 and 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. 

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 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 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 (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 (i.e. 100 mg/m³ for hamsters, unclear for guinea pigs), 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 15 mg/m³ of aluminium powder showed 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 Al2O3 dust (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 chronic toxicity guideline (OECD TG 452) 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.

Cross-reference 5

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity, other

Remarks:

inhalation: dust and Intratracheal injections

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1973

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD TG 452

Deviations:

yes

Remarks:

: number of animals to low (30 instead of 50 per sex and dose); duration only 1 year instead of 2 years.

GLP compliance:

not specified

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Details on test animals or test system 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.

Route of administration:

other: inhalation: dust and Intratracheal injections

Type of inhalation exposure (if applicable):

whole body

Vehicle:

not specified

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 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 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.

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
According to Table 2 of the article -
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 were exposed to 30 mg/m³ of aluminium oxide dust for a year.

Frequency of treatment:

Inhalation:
6 hr/day; 5 days a week

Dose / conc.:

15 mg/m³ air

Remarks:

pyro powder, atomized powder, flake powder

Dose / conc.:

30 mg/m³ air

Remarks:

pyro powder, atomized powder, flake powder

Dose / conc.:

30 mg/m³ air

Remarks:

aluminium oxide

No. of animals per sex per dose:

Inhalation:
14 and 26 guinea pigs were exposed to pyro powder at 15 and 30 mg/m³, respectively.
15 and 19 guinea pigs were exposed to atomized powder at 15 and 30 mg/m³, respectively.
21 and 25 guinea pigs were exposed to flake powder at 15 and 30 mg/m³, respectively.
12 guinea pigs were exposed to aluminium oxide at 30 mg/m³.

Control animals:

yes

Details on study design:

Control animals:
12 guinea pigs were untreated (laboratory controls). Five animals were examined per time point.
Aluminium oxide (numbers and dosing described above) were included as “non-fibrogenic” controls.

Positive control:

No.

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).

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 data.

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not specified

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

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

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 2 to 3 months of exposure in two guinea pigs.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
The authors reported no tumors in the lungs of guinea pigs in the inhalation series.

LUNG HISTOLOGY
Inhalation series:
Al-powders:
All three species developed alveolar proteinosis (AP);
AP that developed was not severe and cleared readily on cessation of exposure. Dust particles were also readily cleared.
Persistent changes: similar to hamsters but altered alveoli contained dust-filled macrophages to a greater extent than hamsters. These effects are observed for treated animals only. Inconsistency between Table 2 and Figure 7 with respect to doses used limit the utility of these results for the extraction of an LOAEC.
No tumors were observed in the guinea pig lungs.

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

>= 30 mg/m³ air

Based on:

test mat.

Remarks:

Al2O3 dust

Sex:

not specified

Critical effects observed:

not specified

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 interspecies 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 Al2O3 content 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 and 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. 

 

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 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 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 (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 (i.e. 100 mg/m³ for hamsters, unclear for guinea pigs), 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 powder showed 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 Al2O3 dust (66% <1 μm) 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.

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

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

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

Test material information

Constituent 1

Reference substance name:

Aluminium powder, aluminium oxide (Al2O3)

IUPAC Name:

Aluminium powder, aluminium oxide (Al2O3)

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 or test system 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 / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/m³ air

Remarks:

pyro powder, atomized powder, flake powder

Doses / concentrations 2

Dose / conc.:

30 mg/m³ air

Remarks:

pyro powder, atomized powder, flake powder

Doses / concentrations 3

Dose / conc.:

50 mg/m³ air

Remarks:

pyro powder, atomized powder

Doses / concentrations 4

Dose / conc.:

100 mg/m³ air

Remarks:

pyro powder, atomized powder

Doses / concentrations 5

Dose / conc.:

30 mg/m³ air

Remarks:

aluminium oxide

Doses / concentrations 6

Dose / conc.:

75 mg/m³ air

Remarks:

aluminium oxide

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 75 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

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

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

1

Dose descriptor:

LOAEC

Remarks:

local effects

Effect level:

15 mg/m³ air

Based on:

test mat.

Remarks:

Al powder

Sex:

not specified

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

>= 75 mg/m³ air

Based on:

test mat.

Remarks:

Al2O3 dust

Sex:

not specified

Basis for effect level:

other: no adverse effect observed at the highest dose tested

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	75	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 the flake 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 75 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 75 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 and powders 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 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 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 (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.   

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 powder showed 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 chronic toxicity guideline (OECD TG 452) 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.