Aluminium oxide

Endpoint summary

Description of key information

chronic toxicity:

oral, rat: NOAEL: 322.5 mg/kg bw/day as aluminium citrate (equivalent to 113.36 mg Al oxide/kg bw/day)

inhalation, rat: NOAEC >= 75 mg/m³ as aluminium oxide

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

short-term, sub-chronic, chronic

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

weight of evidence

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

322.5 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

Al citrate; corresponding to 30 mg Al/kg bw/day

Sex:

male/female

Basis for effect level:

other: neuromuscular effects (hindlimb grip strength, footsplay) at 1075 mg Al citrate/kg bw/day

Remarks on result:

other: woe, source, RA-A, 31142-56-0, Alberta Research Council Inc, 2010

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

Al chloride; corresponding to 180 mg Al/kg bw/day

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects observed at highest dose tested

Remarks on result:

other: woe, source, RA-A, 1327-41-9, Beekhuijzen, 2007

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

80 other: mmol/L

Based on:

test mat.

Remarks:

Al citrate; corresponding to 6-11 mmol Al/kg bw/day

Sex:

female

Basis for effect level:

clinical biochemistry

haematology

Remarks on result:

other: woe, source, RA-A, 31142-56-0, Vittori et al., 1999

Dose descriptor:

NOAEL

Remarks:

systemic toxicity/ local effects

Effect level:

>= 302 other: mg Al/kg bw/day

Based on:

test mat.

Remarks:

Al(OH)3

Sex:

male

Basis for effect level:

other: no adverse effects observed at highest dose tested

Remarks on result:

other: woe, source, RA-A, 21645-51-2, Hicks et al., 1987

Dose descriptor:

NOAEL

Remarks:

systemic toxicity/ local effects

Effect level:

>= 1 034 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

NaAl(SO4)2; corresponding to 103 mg Al/kg bw/day

Sex:

male

Basis for effect level:

other: no adverse effects observed at highest dose tested

Remarks on result:

other: woe, source, 7785-88-8, Katz et al., 1984

Critical effects observed:

not specified

Conclusions:

The available oral repeated dose toxicity studies with the source substances (CAS 31142-56-0, 1327-41-9, 21645-51-2, 7785-88-8) revealed a NOAEL for systemic toxicity in a chronic study of 30 mg Al/kg bw/day with regard to systemic toxicity. Applying the read-across approach, similar results are expected for the target substance (CAS 1344-28-1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

113 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on the presence of a common metal ion, or ion complex including a hydrated metal ion, and following from this a similar chemical behaviour (refer to endpoint discussion for further details).
The available information as a whole is sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

System:

nervous system

Organ:

not specified

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

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

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:

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.

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

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
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.

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.

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.

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

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.

 

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline available

Principles of method if other than guideline:

Although the non-physiologic modes of administration used in this study limit its utility for the derivation of a dose-descriptor, the results clearly show the importance of the physical characteristics of the alumina dust for the biological response. The intratracheal instillation doses overloaded clearance mechanisms in all cases. Only one dose was used. Two species were investigated, animal weights were monitored, cytological and biochemical analyses of BALF were undertaken in addition to histopathological examinations of lung tissue.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ivanovas, Kisslegg, Gremany
- Age at study initiation: 2 month
- Weight at study initiation: 200 - 250 g
- Diet: Standard diet ad libitum
- Water: ad libitum

Route of administration:

other: Intratracheal Instillation

Type of inhalation exposure:

not specified

Vehicle:

not specified

Remarks on MMAD:

MMAD / GSD: No information was provided on the MMAD and GSD. The % of particles with sizes less than 11 μm was reported.
Further detail on particle characteristics:
Additionally, alumina dusts (1) to (5) had particle size distributions similar to those in air of aluminium smelters with prebake pots (smelter-grade aluminas). Alumina dust (6) contained particle sizes unlikely to occur in potroom air. Alumina dust (7) had a particle size distribution typical of the inhalable fraction of potroom dust.

Details on inhalation exposure:

The dusts were administered without prior sterilization in order to minimize any modifications to their physical characteristics that might influence the biological effects. Sterile isotonic saline was injected into a vial containing a pre-weighed sample of dust. The suspension was shaken vigorously by hand and then using a Vortex shaker until the animal was ready for instillation. Rats were lightly anaesthetized with methoxyflurane and instilled intratracheally with 0.1 mL of the fresh suspension or, for the negative controls, sterile saline. The instillation was guided using a laryngoscope.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Two weeks

Frequency of treatment:

5 injections.

Dose / conc.:

50 other: mg

Remarks:

alumina; total dose by 5 injections over a 2 week period

Dose / conc.:

25 other:

Remarks:

quartz; total dose

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

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

Positive control:

Positive control: Quartz (25 mg)
Size: median particle size 4.76 µm
Specific surface BET (m²/g ): 3.1 m²/g

Observations and examinations performed and frequency:

Observations and examinations performed:
Animal weights were recorded as an index of general health.
Bronchoalveolar lavage:
- BAL fluid was obtained by flushing the lungs of the sacrificed animals with 8 to 10 mL of physiological saline. The saline was introduced over the period of a minute, allowed to remain for a minute and then withdrawn. Flushing was carried out twice. The authors reported that 80 to 85% of the lavage fluid was recovered.
The following parameters were determined in the BALF:
- lactate dehydrogenase activity (LDH) in the cell-free supernatant (Bergmeyer and Bernt, pp. 574-579 in Methods of Enzymatic Analysis, Vol. 2, ed. By H.U. Bergmeyer. New York, Academic Press, 1974);
- total protein (method of Bradford, Analyt Biochem 1976; 72: 248-254);
- Cytology: cell counts (using a Neubauer’s hemocytometer chamber) and types (in a “cytocentrifuged stained preparation”).
- Lung tissue pathology (methods reported below).
Frequency of the observations and examinations:
Weights were recorded each week.
Observations were made at 4 timepoints; 60, 90, 180 and 361 days after exposure.

Sacrifice and pathology:

Animals were anaesthetized with sodium-pentobarbital and sacrificed by exsanguination. Blood was aspirated from the peritoneal cavity with a Pasteur pipette.
Histopathological examinations of lung tissue were conducted using sections embedded in paraffin. Five sections were taken; at least one from each lobe that were stained with hematoxylin-eosin and van Gieson (to show connective tissue and collagen fibres).

Other examinations:

A separate experiment was conducted in male, one month old, NMRI mice. The mice were injected intra-peritoneally with a 0.5 mL volume of 1% suspension of dust in sterile isotonic saline. Five mice were sacrificed at 30, 90 and 180 days and 10 mice were examined at 360 days. Nodules formed by the dust on the anterior wall of the abdomen and over the peritoneal viscera were examined macroscopically and histopathology using formalin-fixed, paraffin sections stained with hematoxylin, eosin and also van Gieson stain to identify connective tissue and collagen fibres.

Statistics:

Non-parametric tests were used to compare LDH activity levels and protein content in BALF between the treated groups and the controls.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

for mortality data, no data for clinical signs

Mortality:

mortality observed, treatment-related

Description (incidence):

for mortality data, no data for clinical signs

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Rats (Intratracheal Instillation)
Weight:
The best weight gains were observed for control animals and animals dosed with alumina Dust(2). A relative reduction in weight gain (15%) was observed in animals dosed with quartz or alumina Dust(1).

Cytotoxicity:
The level of LDH activity in the saline control was 38 ± 28 mU/mL. At 60 days and 90 days post-instillation, LDH activity was significantly higher than control (p < 0.05) in all dusts; the highest value was observed in the quartz positive control. At 180 days, the results for Dust (6) were not significantly different than in the controls. For, all other dusts, the LDH activity at days 180 and 360 was significantly higher than control values (p < 0.05). In summary, persistent increases throughout the duration of the experiment were observed for Dust (1) and Dust (3) (ca. 7-8x higher). Increases were also observed for the other dusts, with the exception of Dust (6), but the magnitude was smaller. Dust (6) showed the lowest values (44 – 101) and quartz (positive control) the highest values (20-30x higher than the control). The results suggest the persistence of the inflammatory response in animals dosed with alumina.

Total soluble protein levels in the saline control were 0.19 ± 0.08 mg/mL. Statistically significant increases were observed for all dusts. Dusts (5), (6) and (7) were the lowest (2-4 times higher than the control, Dust(1) was 6x higher at 180 days, Dust (2) & Dust (4) 3-4 times higher and Dust (3) 7 times higher at 360 days. The positive control, quartz, had levels 10-20 times higher

Cytology:
Alveolar macrophages (AM). In the saline control, 98% of all nucleated cells were AM. For Dusts (1) to (7) the AM decreased in percentage, increased in size and many were vacuolated.
Polymorphonuclears were increased in all alumina-treated animals; 10-15% were multinuclear (except Dust (5) which had 3-5% multinuclear). The increase persisted.

Histopathology1 year post ITI:
The histological findings for Dusts (1) to (5) were similar with a mild to severe alveolar reaction, moderate levels of aggregates of dust-laden macrophages but with no collagen. Regional lymph nodes contained dust-laden phagocytes but also no collagen.

In animals instilled with Dust (6), the chemical grade “Aerosil”, an alveolar reaction was almost absent, dust-laden interstitial macrophages were very abundant and collagen was present in considerable amounts. Regional lymph nodes contained dust-laden phagocytes and some collagen fibres.

Animals dosed with Dust (7) showed a mild alveolar reaction, abundant dust-laden interstitial macrophages and some collagen. Some collagen fibres were present in regional lymph nodes in addition to dust-laden phagocytes. The response was intermediate between the response to the smelter-grade alumina and Dust (6), the chemical grade product.

Animals dosed with quartz had a very severe alveolar reaction, with very abundant aggregates of dust-laden interstitial macrophages and abundant collagen. Whorls of collagen were observed in regional lymph nodes.
The saline controls exhibited normal lung pathology.

Mice (intraperitoneal injection)

The macroscopic examination of organs in the animals dosed with aluminas (1) to (5) typically found nodules on the liver, spleen and omentum. The nodules were less numerous in smelter-grade alumina (Dust (1) to (5))-treated animals compared with quartz-treated animals.

Histopathology examinations of nodules showed a progression of responses between quartz (most severe), the Chemical (6) and Lab (7) grade alumina and smelter-grade alumina (Dusts (1) to (5) – least severe). In the animals dosed with Dusts (1) to (5), there were some phagocytes and fibroblasts and cell-free dust agglomerates at 30 days. At 360 days post-instillation, some collagen fibres were found encapsulating the dust but the inflammatory reaction was absent. In the animals dosed with Dusts (6) & (7), at 30 days the response consisted of mostly dust-laden macrophages, some free dust particles; a response consistent with a mild inflammatory reaction. At 90 days there were more fibroblasts and some collagen fibres. At 180 and 360 days, many aggregations showed an acellular central part made of free dust and dense collagen fibres. In the positive control animals dosed with quartz, at 30 days the cells were mostly macrophages with some fibroblasts and PMN. By 90 days, there were some collagen fibres at the core of aggregations, but most nodules were still highly acellular. At 180 and 360 days, collagen fibres were abundant. In summary, exposure to quartz, Dust (6) or Dust (7) resulted in nodules with dense collagen cores. This did not occur on exposure to the smelter-grade aluminas.

Critical effects observed:

not specified

Conclusions:

The results from this study provide clear evidence for an extensive and inflammatory reaction in the positive control (quartz-treated) animals. In contrast, dusts (1) to (5), the smelter-grade aluminas, showed no evidence of a progressive fibrotic effect. Dusts (6) and (7), the chemical and laboratory grade aluminas, respectively, showed evidence of a fibrotic effect. The response for dust (7) was intermediate between the smelter-grade aluminas and the chemical-grade dust (6).

Executive summary:

Ess et al. (1993) investigated the fibrogenic potential of alumina samples from different sources and with different physical properties. The test materials were five fine alumina dusts obtained from sieving raw alumina samples (Dusts (1) to (5)), a chemical grade alumina (Dust (6)) and an alumina produced in the laboratory (Dust (7)). The alumina dusts (1) to (5) had particle size distributions similar to those in air of aluminium smelters with prebake pots. Alumina Dust (6) contained particle sizes unlikely to occur in potroom air and the laboratory grade dust (Dust (7)) had a particle size distribution typical of the inhalable fraction of potroom dust. Two experiments were conducted; one in female Sprague-Dawley rats with administration by intratracheal instillation and the other in male NMRI mice with administration of the dusts by intraperitoneal injection. In the rat study, a total dose of 50mg was administered to the lightly anaesthetized animals using 5 injections of well-shaken suspensions (0.1 mL in sterile isotonic saline) of non-sterilized dust over a two week period. Animal weights were recorded weekly and five animals were sacrificed 60, 90, 180 and 361 days after exposure. Quartz was included as a positive control (total dose: 25 mg) and sterile isotonic saline as a negative control. However, it is not clear whether negative controls were included for every observation time point. Bronchoalveolar lavage fluid (BALF) was collected using two flushes and subjected to cytological and biochemical (lactate dehydrogenase (LDH), total protein) analyses. Histopathological examinations of lung tissue were undertaken staining to show general histology (hematoxylin-eosin) and also connective tissue and collage (van Gieson).

 

Weight gains were highest in control animals and animals dosed with alumina Dust (2). A reduction in weight gain (15%) was observed in animals dosed with quartz or alumina Dust (1). Biochemical analyses of the BALF showed significantly higher LDH activity in treated versus control animals at 60 and 90 days post-instillation for all the dusts with the highest value observed in the quartz positive control. At 180 days, the results for Dust (6) were not significantly different from the controls. For, all other dusts, the LDH activity at days 180 and 360 was significantly higher than control values (p<0.05). In summary, persistent increases throughout the duration of the experiment were observed, most notably for Dust (1)and Dust (3)(ca. 7-8x higher). Statistically significant increases in total protein were observed for all dusts. Dusts (5), (6) and (7) were the lowest (2-4 times higher than the control, Dust (1) was 6x higher at 180 days, Dust (2) & Dust (4) 3-4 times higher and Dust (3) 7 times higher at 360 days. The positive control, quartz, had levels 10-20 times higher. Cytological analyses showed that 98% of all nucleated cells were AM in the saline control. For Dusts (1) to (7) the AM decreased in percentage, increased in size and many were vacuolated. Polymorphonuclears were increased in all alumina-treated animals; 10-15% were multinuclear (except Dust (5)which had 3-5% multinuclear). The increase persisted. The histological findings for Dusts (1) to (5) one year post-intratracheal instillation were similar with a mild to severe alveolar reaction, moderate levels of aggregates of dust-laden macrophages but with no collagen. Regional lymph nodes contained dust-laden phagocytes but also no collagen. In animals instilled with Dust (6),the chemical grade “Aerosil”, an alveolar reaction was almost absent, dust-laden interstitial macrophages were very abundant and collagen was present in considerable amounts. Regional lymph nodes contained dust-laden phagocytes and some collagen fibres. Animals dosed with Dust (7) showed a mild alveolar reaction, abundant dust-laden interstitial macrophages and some collagen. Some collagen fibres were present in regional lymph nodes in addition to dust-laden phagocytes. The response for Dust (7) was intermediate between the response to the smelter-grade alumina and dust (6), the chemical grade product. Animals dosed with quartz had a very severe alveolar reaction, with very abundant aggregates of dust-laden interstitial macrophages, interstitial nodules and abundant collagen. Whorls of collagen were observed in regional lymph nodes. The saline controls exhibited normal lung pathology.

  

The mice were injected intra-peritoneally with a 0.5 mL volume of 1% suspension of dust in sterile isotonic saline. Five mice were sacrificed at 30, 90 and 180 days and 10 mice at 360 days. Nodules formed by the dust on the anterior wall of the abdomen and over the peritoneal viscera were examined macroscopically and histopathology using formalin-fixed, paraffin sections stained with hematoxylin, eosin and also van Gieson stain to identify connective tissue and collagen fibres. Nodules were observed typically on the liver, spleen, and omentum of the treated mice. The nodules were less numerous in smelter-grade alumina (Dust (1) to (5))-treated animals compared with quartz-treated animals. Histopathology examinations of nodules showed a progression of responses between quartz (most severe; progressive fibrotic), the chemical-grade Dust (6) and laboratory grade Dust (7) and then the smelter-grade alumina (Dusts (1) to (5) – least severe).

 

Overall, all dust samples produced an inflammatory alveolar reaction on intratracheal instillation at these doses. The smelter-grade dusts, dusts (1) to (5) did not show evidence for a fibrotic effect in the rats’ lungs during the period of a year following intratracheal instillation. In contrast, Dust (6), the chemical grade, ultrafine non-alpha alumina and Dust (7), the laboratory grade alumina, showed evidence of definite fibrotic changes. The doses that were instilled into the rats overloaded clearance mechanisms – yet there was a difference in intensity of response between the dusts.

Although the modes of administration used in this study limit its utility for the derivation of a dose-descriptor, the results clearly show the importance of the physical characteristics of the alumina dust on the biological response. The intratracheal instillation doses overloaded clearance mechanisms in all cases. Only one dose was used. Two species were investigated, animal weights were monitored, cytological and biochemical analyses of BALF were undertaken in addition to histopathological examinations of lung tissue. The study was well-reported, described the test-items and methods adequately providing reliable but non-guideline information useful for the risk assessment.

Reference 3

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.

Reference 4

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.

Reference 5

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

3 mg/m³ air

Based on:

test mat.

Remarks:

AlO(OH)

Sex:

male

Basis for effect level:

other: overall effects cytology and biochemical parameters in lavage fluid

Remarks on result:

other: supporting, source, RA-A, 24623-77-6, Pauluhn, 2009a

Conclusions:

The available oral repeated dose toxicity studies with the source substances (CAS 24623-77-6) revealed a NOAEC for local effects of 3 mg/m³ air AlO(OH)/kg bw/day. Applying the read-across approach, similar results are expected for the target substance (CAS 1344-28-1).

Reference 6

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1993

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Documentation is insufficient for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

Cytology results and analysis of biochemical markers in bronchoalveolar lavage fluid obtained from rats after intratracheal instillation of test materials can provide useful information on the inflammatory state of the lung tissue and also the likelihood of long-term effects due to changes in the extra-cellular matrix. The results are informative for possible mechanisms of action and if, substances with better known modes of action e.g. silica, stereotypical low cytotoxicity poorly soluble particles, are also tested concurrently can be useful for ranking inflammatory and fibrogenic potential of materials.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

not specified

Details on test animals or test system and environmental conditions:

Details on test animals and environmental conditions:
The rats with an initial weight of 200 g were kept in litters of at most four animals. No other information on environmental conditions is provided.

Diet and water:
Unlimited access to food and water. No information on the type of diet.

Acclimation and monitoring animal health:
No details were provided on acclimation or monitoring of animal health.

Route of administration:

other: Intratracheal instillation

Type of inhalation exposure:

not specified

Vehicle:

not specified

Remarks on MMAD:

MMAD / GSD: No information was provided in the aerodynamic sizes of the particles. Electron-microscope determined particles sizes were provided in the article (see above in the Details on Test Materials Section).

Further detail on particle characteristics (e.g. shape):
SA contained a small number of Al2O3 fibres 0.05 µm wide and 1-3 µm long.

Details on inhalation exposure:

Instillation was performed under ether anaesthesia. The rat was fixed in a semi-upright position. A cannula was passed through the mouth into the trachea via the larynx. A 40 mg alumina sample was suspended in 0.5 ml saline and injected into the trachea.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Single instillation.

Frequency of treatment:

Single instillation.

Dose / conc.:

40 other: mg

Remarks:

equivalent to 200 mg/kg bw for rats weighing 200 g

No. of animals per sex per dose:

Twenty one rats were instilled with PA and 21 rats with SA.

Control animals:

yes, concurrent vehicle

Details on study design:

The rats were killed 1, 4 and 12 months after the instillation. Seven rats exposed to PA and seven rats exposed to SA were killed at each time point. Five control rats were killed at 1 month, five at 4 months and seven at 12 months after the instillation.
Histopathological examination of the lung tissue was conducted on 5 animals in each time and treatment group.

Positive control:

No.

Observations and examinations performed and frequency:

Bronchoalveolar lavage:
The trachea was cannulated and 5 ml of Hank’s balanced salt solution at 37 °C was infused into the lungs at hydrostatic pressure of 20 cm H2O. Three minutes later, the fluid was drained by gravity into a cooled siliconized tube. The flushing procedure was repeated 10 times.
The following parameters were determined in the BAL fluid:
- Total cell count (in a Burker chamber)
- Differential cell count was performed on cytosentrifugal smears prepared in a Cytospin 2 apparatus at 500 rev/min for 3 min. Cells were stained by the May-Grunwald Giemsa method; 500 cells were counted.
- Albumin concentration was determined by rocket immunoelectrophoresis (Laurell, C. B. Electroimmuno assay. Scand J Clin Lab Invest Suppl. 1972; 124:21-37).
- Fibronectin concentration was determined by a double sandwich enzyme linked immunosorbent assay (Blaschke, E.; Eklund, A., and Hernbrand, R. Determination of fibronectin and its degradation products in bronchoalveolar lavage fluid. Scand J Clin Lab Invest. 1990; 50(6):619-625)
- Hyaluronan concentration (according to Engstrom-Laurent, A.; Laurent, U. B.; Lilja, K., and Laurent, T. C. Concentration of sodium hyaluronate in serum. Scand J Clin Lab Invest. 1985 Oct; 45(6):497-504)
Lung tissue pathology (methods reported below).

Frequency of the observations and examinations:
No information

Sacrifice and pathology:

Animals were killed with ether.
Tissue samples were taken from three different levels of the right lower lobe of the lung, fixed in 10% neutral formaldehyde, embedded in paraffin, and sections examined by light microscopy after staining (hematoxylin eosin).

Statistics:

Student’s t-test for statistical significance of differences between the alumina exposed and control rats at each time point.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

Recovery of the instilled fluid and cell viability (determined by Trypan blue exclusion): no difference between the alumina exposed (PA and SA) and control groups at any time after the instillation
Histological findings in the lung: no difference between alumina exposed groups (PA and SA) and control at any time after the instillation. No signs of fibrosis in any animal.
Total cell count in the BAL fluid
Rats exposed to PA: no significant difference from control rats at any time after the instillation.
Rats exposed to SA: significant increase (p<0.05) compared to control 1 and 12 months after the instillation.
Differential cell count in the BAL fluid
Rats exposed to PA: significantly lower (p<0.01) number of lymphocytes compared to control rats 1 month after the instillation. No difference in the number of macrophages and neutrophils at any time.
Rats exposed to SA: significant increase in the number of macrophages (p<0.05) and neutrophils (p<0.01) 1 and 12 months after the instillation. No significant difference with the control rats in the number of lymphocytes.
Albumin concentration in the BAL fluid
Rats exposed to PA: significantly lower (p<0.05) concentration 4 months after exposure.
Rats exposed to SA: no significant difference from the control rats at any time after exposure
Hyaluronan concentration in the BAL fluid
Rats exposed to PA: no significant difference from the control rats at any time after exposure
Rats exposed to SA: no significant difference from the control rats at any time after exposure
Fibronectin concentration in the BAL fluid
Rats exposed to PA: significant increase 12 months after exposure (p<0.001)
Rats exposed to SA: significant increase 12 months after exposure (p<0.001)

Critical effects observed:

not specified

Conclusions:

This study shows that intratracheal instillation of SA induces early changes in alveolar cell populations indicative of a low-level inflammatory response. The absence of an increase in the number of cells after instillation of PA suggests that fluorides are essential for this type of reaction. The increase in fibronectin concentration 12 month after exposure to both PA and SA suggests that these exposures may lead to a change in the extracellular matrix in rats and that alumina particles, not fluoride is responsible for this effect. However, no fibrosis was observed on histopathological examination of the lung tissue at any time after exposure to either PA or SA. The authors point out that the non-physiological route of administration may cause a pulmonary overload; and thus would also lead to a more severe chronic irritative effect than would occur under exposures by inhalation. It cannot be concluded from the results of this study whether the increases in fibronectin are an effect specific to the chemical substance alumina or are due only to the presence of retained particulate matter i.e.”inert” dust.

Executive summary:

Tornling et al. (1993) investigated the longer-term effects on histology and biochemical and cellular parameters in BALF from rats exposed by single ITI to primary potroom aluminium oxide (without adsorbed fluorides - PA), secondary potroom alumina (post-fluoride adsorption - SA) or saline. A single dose of 40 mg, equivalent to 200 mg/kg bw for rats weighing 200 g, was administered in 0.5 mL saline. Albumin, fibronectin and hyaluronan were measured in the BALF. Seven rats were sacrificed at 1, 4 and 12 months post-instillation. PA-treated rats showed no significant increase in total cells, macrophages, lymphocytes or neutrophils compared with the saline control at any time point. SA-treated rats showed significantly elevated numbers of macrophages and neutrophils compared to the control animals at one and 12 months indicative of a low-level inflammatory response. . The absence of an increase in the number of cells after instillation of PA suggests that fluorides are essential for this type of reaction. Significantly elevated fibronectin levels at 12 months were observed in both alumina-treated groups, suggesting that these exposures may be associated with a change in the extra-cellular matrix leading to a fibrotic response in rats. Histological sections, however, showed no signs of fibrosis in this single-dose study. The authors point out that the non-physiological route of administration may cause a pulmonary overload; and thus would also lead to a more severe chronic irritative effect than would occur under exposures by inhalation. It cannot be concluded from the results of this study whether the increases in fibronectin are an effect specific to the chemical substance “alumina” or are due only to the presence of retained particulate matter i.e.”inert” dust.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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

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:

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.

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

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
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.

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.

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.

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

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.

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

75 mg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

There are no studies available on the repeated dose toxicity of aluminium oxide by the oral route.

In terms of hazard assessment of toxic effects, available data on the repeated dose toxicity of other aluminium compounds was taken into account by read-across following a structural analogue approach, since the pathways leading to toxic outcomes are likely to be dominated by the chemistry and biochemistry of the aluminium ion (Al³⁺) (Krewski et al., 2007).

A detailed rationale and justification for the analogue read-across approach is provided in the technical dossier (see IUCLID section 13).

A GLP study was performed using aluminium chloride basic in accordance with OECD Test Guideline (TG) 422 "Combined Repeated Dose and Reproductive/Developmental Screening Test" (Beekhuijzen 2007). No mortality or clinical signs of intoxication were observed in male and female Wistar rats due to treatment with Al chloride basic at dose levels of 40, 200, and 1000 mg/kg body weight which contribute 0, 7.2, 36 and 180 mg Al/kg bw/day, respectively.

Treatment with Al chloride basic by oral gavage revealed paternal toxicity (irritation effect on glandular stomach mucosa, local effect) at 1000 mg/kg in both the male and female Wistar rats. Based on findings observed macroscopically (red foci or thickening of the grandular mucosa of the stomach) and supported by microscopic examination, the maternal/parental No Observed Adverse Effect Level (NOAEL) for local toxic effects on stomach was established at 200 mg/kg and LOAEL – at level 1000 mg/kg, for both males and females.

Several statistically significant changes in clinical biochemistry parameters were observed at 1000 mg/kg: decreased Hb level in males, MCHC in both Al treated males and females, decreased alkaline phosphatase activity, decreased total protein and albumin levels in blood serum and increased potassium level. Decreased Hb levels were observed in two other doses in males but no dose response relationship was observed. Lack of relevant baseline values for the observed clinical data limit the interpretation of the results.The authors consider clinical biochemistry and haematology changes observed at 1000 mg/kg to be of slight nature and generally within the range expected for rats of this age and strain.Because any morphological correlates were absent, these changes were considered not indicative of organ dysfunction and not of toxicological significance.

No reproduction, breeding and early post-natal developmental toxicity was observed in rats at 1000 mg/kg body weight for males and females. Based on the reported results, a NOAEL for reproduction, breeding and early post-natal developmental toxicity was suggested at a level of 1000 mg/kg bw, the highest dose tested in this study.

 

In another study (Hicks et al., 1987) Male Sprague-Dawley rats were exposed to aluminium-compounds with diet. The animals were randomly assigned to five groups, 25 animals in each. The groups received 1) basal diet (control), 2) aluminium hydroxide (302 mg Al/kg body weight), 3) KASAL -the basic form of sodium aluminium phosphate containing ≈6% of Al (141 mg Al/kg body weight), 4) KASAL II - the basic form of sodium aluminium phosphate containing ≈13% of Al (67 mg Al/kg body weight) and 5) KASAL II (288 mg Al/kg body weight). Treatment continued for 28 days, during which the animals were observed twice daily for their behaviour, signs of toxicity, and mortality. General physical examinations, body weight and food consumption measurements were performed weekly. After 28 days of treatment, 15 animals from each group were killed. Blood was collected from 5 rats of each group for blood cell counts, haemoglobin concentration, haematocrit and serum chemistry measurements. These rats were subjected to gross necropsy and histopathological examination. Femurs from 10 rats were taken for possible aluminium analysis; femurs from 5 rats were analyzed for Al concentrations. Five rats were allowed to recover for 2 months and five rats for 5 months after termination of the treatment. During these recovery periods, the rats received the basal diet and were observed daily; body weight and food consumption were measured monthly. Femurs were collected at autopsy from these rats for aluminium analysis. During the entire experimental period, no mortality was reported and no treatment-related clinical signs were observed. All clinical observations were characteristic of male Sprague-Dawley rats of relevant age. There were no significant group differences in body weight, food and water consumption and haematological parameters.A mild (2 - 4%) but significant increase in serum sodium level was observed in all treated animals.However, all increased sodium levels were within the range of historical control for rats of the same age in the laboratory. A significant 16% increase in absolute kidney weight was reported in the group of rats receiving KASAL II at 67 mg Al/kg bw. This increase appeared not to be treatment-related because no such increase was seen in the group of animals treated with this substance at 288 mg Al/kg bw. There were no other significant group differences in organ weights. All lesions seen at microscopic examination were “those normally expected for young adult male Sprague-Dawley rats” (Hicks et al., 1987). No lesions suggestive of a treatment-related effect were seen.Aluminiumconcentrations in all femur samples from all groups were < 1 ppm and most were below the limit of detection or quantification.The distribution of samples in which Al was not detectable, was detectable but not quantifiable or was quantifiable, was similar in all the groups. It should be noted that this comparison was based on small numbers of samples from each group (5). Al was quantifiable in all 5 samples from animals treated with Al(OH)3, in 2 samples from the control animals and in none of the samples from animals treated with KASAL or KASAL II. The results of this study provide no evidence for significant deposition of Al in the bone and no evidence for adverse effects induced by Al hydroxide or basic food grade sodium aluminium phosphate (KASAL and KASAL II) during 28-day dietary administration at daily doses up to ≈300 mg Al/kg body weight. 

 

Sodium aluminium phosphate was administered to beagle dogs with diet at concentrations 0% (control), 0.3%, 1.0% and 3.0% for 6 months (Katz et al., 1984). There were no significant group differences in body weight throughout the experiment. Reductions in mean body weight occurred in all groups during week 27, which the authors attributed to “pretermination tests and increased handling by technicians.” No treatment-related clinical signs and no ocular changes in any of the animals were observed. In most weeks, treated male and female dogs consumed less food than control dogs. In male animals, none of the differences in mean food consumption values was statistically significant. In females, significant reductions occurred “sporadically”. The authors did not consider these differences in food consumption as “toxicologically significant”, the conclusion that was supported by the absence of corresponding reduction in body weights. The treatment did not have any effect on haematological and blood biochemistry parameters, urinalysis results and results of analysis for occult blood in faeces. There were no significant differences in mean organ weights between the treated groups and the control group. Gross pathology and histopathology findings were in the “normal range of variations for dogs of this strain and age”; no treatment-related lesions were observed. The results of this study provide no evidence for toxicity of acidic form of sodium aluminium phosphate during 6-month administration at concentrations up to 3% in the diet.

 

Aluminium citrate was administered to ten female Sprague Dawley rats with drinking water at a concentration of 80 mmol/L for 8 months (equivalent of 6 - 11 mmol Al/kg bw/day) (Vittori et al. 1999). Plasma iron concentration and total iron-binding capacity were not different in the control and the Al treated rats, indicating that the Al treated animals were not depleted of iron. There were no significant group differences in blood urea concentration, which suggests that kidney function was not altered by Al administration. Significantly lower haematocrit and blood Hb concentration were observed in the Al treated rats than in the control rats. Significantly higher reticulocyte count, abnormal erythrocyte morphology, a significant inhibition of (late colony-forming unit-rethroid, CFU-E) growth and a significant reduction of 59Fe uptake in the bone marrow were reported in the Al treated rats. Plasma haptoglobin concentration was significantly lower in the Al treated animals than in the control animals. This and the presence of abnormal erythrocytes in the Al treated rats are indicative of intravascular haemolysis. Scanning electron microscopy combined with EDAX detected Al inside circulating erythrocytes with abnormal shape from animals in the Al treated group. Al concentrations in the bone, spleen, liver, kidney and plasma were significantly higher in the Al treated group than in the control group. No significant group difference in brain Al concentrations was seen. There was no correlation between plasma Al concentrations and Al levels in the organs or any other biochemical data. The results of this study suggest that Al may affect erythropoiesis in rats with normal renal function.

 

A recent combined one-year developmental and chronic neurotoxicity study with Al-citrate (Alberta Research Council Inc, 2010) may be of interest for the evaluation of the neurotoxicity of aluminium hydroxide, aluminium metal and aluminium oxide taking into consideration the tenfold lower bioavailability of aluminium hydroxide, aluminium metal and aluminium oxide compared to Al-citrate and excluding effects that can likely be related to the salt rather than the cation. The study was conducted according to OECD TG 426 and GLP, and the exposure covered the period from gestation day 6, lactation and up to 1 year of age of the offspring. Pregnant Sprague-Dawley dams (n=20 per group) were administered aqueous solutions  via drinking water of  3225 mg/Al citrate/ kg bw/day (300 mg Al/kg bw/day); 1075 mg/Al citrate/kg bw/day (100 mg Al/kg bw/day); 322.5 mg/Al citrate/kg bw/day (30 mg Al/kg bw/day). The highest dose was a saturated solution of Al-citrate. Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L, equimolar in citrate to the high dose Al-citrate group) or plain water (control group). The Al citrate and Na-citrate were administered to dams ad libitum via drinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Dams were sacrificed at PND 23. At PND 4  1 male and 1 female pup of each litter  were allocated to 4 testing groups: D23-sacrifice group for pre-weaning observations and D23 neuropathology, D64, D120 and D365 postweaning groups for post weaning observations and neuropathology at the respective days of sacrifice. Endpoints and observations in the dams included water consumption, body weight, morbidity and mortality and a Functional Observational Battery (FOB) (GD 3 and 10, PND 3 and 10). Pups were examined daily for morbidity and mortality. Additional neurobehavioral tests were performed at specified intervals and included, T-maze, Morris water maze, auditory startle, and motor activity. Female pups were monitored from PND26 for vaginal opening, male pups from day 35 for preputial separation. Clinical chemical and haematological analysis was performed for each group on the day of scheduled sacrifice. Al-concentrations were determined in blood, brain, liver, kidney, bone and spinal cord tissues by inductively coupled plasma mass spectrometric analysis. Further metals such as iron, manganese, copper and zinc were also determined. The pathological investigation includes rain weight and neuropathology. Statistical analyses were performed using the SAS software release 9.1. Data collected on dams and pups were analysed separately. All analysis on pups was performed separately for each sex. Statistical significance was declared from P ≤ 0.05.

Results: Dams: Eight high dose dams developed diarrhoea. In the Na-citrate group one dam stopped nursing and the pups were euthanized. No significant differences between mean body weights of dosed animals compared to controls were observed during gestation and lactation. During gestation and lactation low and mid dose group animals consumed considerably more fluid than controls and high dose group animals. This is not considered treatment related as there was no dose response. In all animals the target dose was exceeded during lactation due to the physiologically increased fluid consumption.

Pups: During the pre-weaning phase weights of mean body weights of male and females in the sodium citrate and high dose group were significantly lower than the untreated controls. This suggests a citrate rather than Al-related effect. No differences between treated and control animals were observed in the FOB. No other clearly treatment related effects were observed pre-weaning.

F1-postweaning: General toxicity

No significant differences in body weights throughout the study were observed between low and mid-dose animals sodium-citrate and untreated controls. High dose males had significant lower body weights than controls by PND 84. These animals also had clinical signs. At necropsy urinary tract lesions were observed in the animals of the high dose group, most pronounced in the males, hydronephrosis, uretal dilatation, obstruction and/or presence of calculi. All high dose males were sacrificed on study day 98. The effect is probably due to Al-citrate calculi precipitating in the urinary tract at this high dose level. This effect is related to the citrate salt and cannot be attributed to the Al-ion. Female high dose animals showed similar urinary tract lesions, but with a lower incidence and severity. Urinary tract lesions were also observed in single mid dose males, but also in a few sodium citrate and control animals. Fluid consumption during the study was increased in the sodium citrate and Al-citrate groups (in particular high and mid dose) compared to controls. This is probably due to the high osmolarity of the dosing solutions. However, the consumed dose levels decreased in all dose groups during the study. In the beginning the target dose was considerably exceeded, while versus the end of the study it was considerably below the target dose.  According to the authors the assigned dose levels still remain valid.

Developmental landmarks:

In sodium citrate controls and high dose males and females the number of days to reach preputial separation or vaginal opening was longer than in untreated control animals. This may be related to the lower body weights in these animals at the respective time-point. As the sodium citrate group showed similar retardation this effect cannot be allocated to the aluminium cation.

Neurobehavioral testing

No consistent treatment related effects that could be related to Al-ion exposure were observed in the FOB. No treatment related effects on autonomic or sensimotoric function were observed in the study. A weak association between Al exposure and reduced home cage activity, a very weak association with excitability, some association with neuromuscular performance were reported but according to the authors this may also be related to group differences in body weight, and an association with physiological function and is thus not considered clearly treatment related. No treatment related effect on general motor behavior was observed. No clearly treatment related effect on auditory startle response was observed. There was no evidence of any treatment related effect on learning and memory in the Morris Water Maze test and no clearly treatment related effects in the T-maze test. Hind limb grip strength and to a lesser extend foot splay were reported to be reduced compared to controls in high and mid dose male and female animals, more pronounced in younger than in older  rats. However, the observed effects can be related to the lower body weights of the individual animals undergoing this test. No details on the individual findings and historical control data are available. It can therefore not be concluded with certainty that the observed neuromuscular effects are primary effects of the treatment and attributable to Al3+. The NOAEL was reported based on this effect as 30 mgAl/kg bw in a conservative approach.

Haematology: No clinically significant differences in hematology were observed at the investigation on day 23. In day 64 and 120 females and day 64 males the high dose group showed slight reduction in hematocrit (males only), mean hemoglobin and mean corpuscular cell volume. No such changes were observed in the 364 day group.

Clinical chemistry: while a number of borderline statistically significant changes were observed, such as globuline levels, alkaline phosphatase and glucose in the high dose group little or no biological significance is associated with them. Elevated creatinine and urea levels in Day 64 males are consistent with the renal toxicity observed in these animals.

Organ weights: Brain weights did not differ among the groups, with two exceptions in the day 64 group males brain weights were significantly lower than controls. In the 120 day female high dose group brain weights were also significantly lower than controls. These findings were not reproduced at the other sacrifice times. Brains to body weight ratios were not significantly different and the lower brain weights can be attributed to the body weight.

Pathology: The main pathology findings were the renal lesions with precipitates in the urinary tract and secondary lesions such as hydronephrosis and uretal dilatation   in particular in the high dose group males and to a lesser extend females. Fluid colonic content was also observed in some high dose animals, in particular males. According to the authors the test item clearly precipitated in the urinary tract causing stone formation and blockage and resulted in fluid colonic content. No other macroscopic effects were observed in other organs.

Histopathology: No treatment related histopahological effects were observed in the nervous system at any time point.

Aluminium concentrations in different organs were dose related. Tissue concentrations were highest in blood, and then in decreasing order brainstem, femur, spinal cord, cerebellum, liver cerebral cortex.

A conservative NOAEL of  322 mg Al-citrate/kg bw  corresponding to 30 mg Al/kg bw was derived from this study (with a bioavailability correction this would correspond to ca. 300 mg Al from Al(OH)3).

The most important effects were however related to a precipitation of the citrate in the kidneys and urinary tract and this effect is not related to the Al3+ ion.  The effects on grip strength and foor splay observed can also not be attributed unequivocally to Al-exposure as they may have been secondary to the general toxicity and body weight differences between treated and control animals undergoing this test.

 

 

 

Inhalation:

Human Studies

Aluminium powder

The majority of published human studies of lung effects on exposure to aluminium powder were conducted prior to 1970 (Krewski et al., 2007 (review); Doese, 1938; Goralewski, 1939 to 1948 in Perry, 1947; Koelsch, 1942; Meyer and Kasper, 1942a,b; Crombie et al., 1944; Mitchell et al., 1959, Mitchell et al., 1961; McLaughlin et al., 1962). Effects were associated with intermittent use of a more permeable and possibly biologically active petroleum-based mineral oil coating in place of stearine (Dinman et al., 1987). The small, cross-sectional study (n = 62) by Kraus et al. (2006) provides some evidence for the development of lung pathology (small, round opacities in the upper lung; a thickening of the interlobular septae) consistent with alveolitis without fibrotic activity on exposure to aluminium powder (respirable size range; with diameters smaller than 5μm). Exposure duration in this study ranged from 78 to 360 months. Multivariate logistic regression showed a significant independent association between Al levels in urine and the occurrence of abnormal high resolution computed tomography (HRCT) findings (OR = 1.008, 1.002 - 1.013; 95% CI; p < 0.006; with adjustment for age, time of exposure, smoking habits, vital capacity, FEV1/VC, and resistance). 

 

“Aluminium” dust

Miller et al.(1984) observed pulmonary alveolar proteinosis in a 44-year old male who had been exposed to high levels of aluminium-containing dust during 6 years as a rail grinder. A recent report (Cai et al., 2007) reported granulomatosis lung disease in a 50-year old woman who had worked in a metal reclamation factory and been exposed to high levels of aluminium dust. Energy dispersive X-ray analysis of the granuloma tissue showed high concentrations of aluminium. Separation of an effect specific to aluminium from an effect due to high doses of dust, or in fact, aluminium oxide, is not possible based on these studies.

 

 

Aluminium smelters - occupational asthma

Donoghue et al. (2010) studied occupational asthma among employees in Al pre-bake smelters of Australia and New Zealand from 1991 to 2006 and examined relations between asthma in highly exposed workers and potroom air contaminants. The authors collected asthma incidence each year by a survey of seven Al smelters using diagnostic criteria developed in 1990 by the Australian Aluminium Council Health Panel. Regular medical surveillance, including respiratory questionnaires and spirometry, was conducted at all smelters with intervals from 3 months to 2 years between examinations depending upon job type and duration of employment. No information was available on ages of the workers, gender or range of length of employment. Asthma cases were identified by surveillance following development of symptoms or a few of the cases were diagnosed by a family physician. Pre-placement criteria and assessment of individual suitability for jobs with exposure to potroom dust, fumes, and gases were introduced before the study period; these criteria evolved over the course of the study and these criteria were not uniform at all smelters. These parameters included a history of asthma beyond childhood, reduced forced expiratory ratio (FER) and evidence of reversible airway obstruction. In some smelters, assessment of non-specific bronchial hyper-responsiveness using methacholine challenge was performed. Incidence rates for occupational asthma were calculated for each smelter and for all smelters combined and the data were presented for each year of the study. All cases of occupational asthma identified among smelter employees (regardless of job category) were divided by the total number of smelter employees (regardless of job category) and the incidence rates were expressed as the number of cases per 1,000 employees per year. These annual surveys also obtained data on the work areas in which asthma cases were reported, but due to limited data on employees in each work area, incidence rates by work area were not calculated. Employees who worked ‘‘in close proximity to pot fume or bath material for several hours a week as part of their normal job” (e.g., potrooms, potroom services, rodding, potlining, cryolite recovery, scrubbing, and alumina) were defined as the highly “bath exposed” workers. Exposure data were based on personal sampling of inhalable particulate, respirable particulate, particulate fluoride (F), gaseous hydrogen fluoride (HF) and total F for potroom employees charged with anode changing as it was the most consistent job across all of the smelters. Exposure data were collected from the breathing zone of potroom employees (the numbers of employees were not provided) under the supervision of qualified occupational hygienists for each year (1996 – 2006), but the study design was such that use of personal respiratory protection was not taken into account.

The statistical significance of changes in exposure concentrations (mg/m³) of inhalable particulate, respirable particulate, particulate fluoride, gaseous hydrogen fluoride, total fluoride across all Al smelters during the study period was assessed by regression P-values and Spearman’s correlation coefficients were calculated for correlations between the incidence rate and each exposure variable. A total of 329 cases of occupational asthma were identified and the highest rate occurred in 1992 (9.46/1,000 per year), but this declined to 0.36/1,000 per year in 2006. This amounted to a 96.2% reduction in asthma incidence. Of the 329 cases, 180 (55%) occurred in potroom production employees and of the total at least 243 of those cases (74%) occurred in employees who were assigned duties in the ‘‘bath exposed’’ areas.The mean proportion of all employees who were ‘‘bath exposed’’ over the period 1991 – 2006 was 50% (2,916/5,827) (no further details provided). The median values of the geometric mean concentrations of inhalable particulate, respirable particulate, particulate F, gaseous HF and total F across all seven of the Australian and New Zealand smelters in the worker’s breathing zone declined over the study period. Statistically significant correlations were observed between the reductions in the incidence rate of asthma and reductions in total respirable particulate, total F, particulate F and gaseous HF. The correlation coefficient was greatest for total F (rs= 0.497).

The Donoghue et al. (2010) results demonstrate reductions in occupational asthma among employees of seven New Zealand and Australian Al smelters from 9.46 per 1000 employees per year in 1991 to 0.36 per 1000 employees per year in 2006. Moreover, this reduction was correlated with reductions in the geometric mean of total F in the breathing zone among employees undertaking anode changing (rs = 0.497, p < 0.001). 

A number of the potroom exposure control measures were implemented during the study period (1991 - 2006) and these included an increased focus on standardized work practices, exposure monitoring programs were improved, hooding of pots was performed, ore and fluoride delivery systems were enclosed and enclosed overhead crane cabins with air purification were added and quality control on anode manufacture was improved to reduce replacement of failures. In addition, crucible cleaning and maintenance operations were isolated from potrooms, anode butt cooling was conducted in areas remote from the main potroom aisleway (a practice employed in only some smelters), exhaust ventilation of pots and forced draft ventilation were added in some smelters, natural dilution ventilation of potrooms was augmented by automated fume control in some smelters, an increased focus on consistent alumina/bath anode covers with no holes in the crust, improved process control to minimize anode effects, reduced process upsets that required intervention, improved fume system maintenance to minimize fume system outages, real-time gaseous HF fluoride monitoring in potroom roves with warning signals, controlled sweeping for fugitive dust on floors (e.g., prohibitions on compressed air for sweeping), automated anode butt cleaning and an increased focus on housekeeping. Among the improvements were: additional education on potential health impacts and work practices to minimize exposures, mandatory respiratory protection with clear rules, introduction of enhanced respirator selection and use of powered air purifying respirators (PAPR), quantitative respirator fit testing, education on respirator use, dedicated respirator maintenance and cleaning centers in some smelters and biological monitoring of urinary F for assessment of respiratory protection in some smelters. The Al smelting process was improved over time by blending low sulfur coke with normal coke to reduce SO2 emissions.

There is no question that misclassification of workers can influence the outcome of these types of studies be they prospective or retrospective in nature. Concern can also arise regarding comparisons based on incomplete or highly variable exposures within jobs or between all workers with asthma. If in these circumstances only “bath exposed” workers are considered, this selection can introduce non-differential misclassification of equal or perhaps even greater gravity to the use of area sampling data or failure to measure workplace levels at all.

Fluoride exposure has previously been associated with development of asthma symptoms and non-specific bronchial hyper- responsiveness in cohort studies of Norwegian potroom workers (Kongerud et al., 1991; 1994; Søyseth et al., 1994). However, occupational exposure data for these older studies were incomplete. The strongest epidemiological evidence is likely to come from an inception cohort study of new workers with detailed longitudinal characterization of personal exposures to the host of airborne materials found in Al smelters (Abramson et al., 1989).

Strengths of the Donoghue et al. (2010) study design include: consistent diagnostic criteria that were applied throughout the study period, prospective collection of asthma incidence and collection of personal samples for the highly exposed workers. While the results are most encouraging, the study does suffer from a number of limitations including: data on cases of occupational asthma come from different sources (onsite medical centers and family physicians and it is not clear whether family physicians applied the same criteria to diagnose occupational asthma); the proportion of cases diagnosed by family physicians is not reported; missing data on numbers of asthma cases and/or numbers of employees for some years; the pre-placement criteria were different at different smelters and “evolved during the study period”; the pre-placement criteria applied only to jobs with highest potential for potroom exposures but the incidence rates of occupational asthma were calculated for all employees regardless of job category; there was no description of date(s) when pre-placement examinations were introduced in smelters and the possible impact of worker reassignment, migration out or replacement of the “bath-exposed” workers on the incidence rate was not discussed; lack of data on potential confounding factors (employee turnover rates, age, tobacco consumption). Although it is not clearly stated whether the mean Al exposures for anode changers represent full-time shift measurements or whether it represents the highest short-term or transient peak exposures to dust and gas, the values are likely to be 8-hour time-weighted-averages (the usual way in which occupational exposures of this type are reported). There may be concern regarding correlations between exposure among the most highly exposed employees and the overall rate of occupational asthma given that no data on the asthma rates among the highly exposed workers were presented. The limitations in exposure measures and failure to account for worker migration out of the industry provide the basis for a Reliability Score of 2. In any case no conclusion on a possible effect of Al-exposure can be drawn from this study.

 

Aluminum smelters exposure to airborne contaminants and respiratory outcomes  

Abramson et al. (2010) investigated the relationships between occupational exposures to airborne contaminants (total F, gaseous fluoride, sulphur dioxide (SO2), coal tar pitch volatiles (as benzene soluble fraction (BSF), oil mist and total inhalable dust) and changes in respiratory function over time. Following a cohort employed at two Australian Al smelters where 446 new employees (77% of the 583 eligible workers) were examined at regular intervals over a period of 9 years (from 1995 to 2003), all participants completed an interviewer-administered questionnaire, pulmonary function tests and skin prick testing for common aeroallergens. Most of the workers were under 35 years old, with a median age for men of 30 (IQR 23-36) years and women of 31 (IQR 25-37) years. At baseline interview, wheeze and chest tightness were reported by 22.6% and 10.6% participants, respectively, and 9.4% were diagnosed with asthma. Baseline pulmonary function findings were within normal values, but 57% were atopic on skin prick testing. At smelter A and smelter B, the proportion of current male tobacco smokers was 32% and 23%, respectively, and the proportions of current female smokers were 39% and 28%, respectively. The proportion of former (19%) and never smokers (51%) was similar between sites.

A task exposure database (TED) was used by site industrial hygienists to record routine air monitoring for the airborne fluorides, SO2, coal tar pitch volatiles (as BSF), oil mist and inhalable dust. Based on these data, a Task Exposure Matrix (TEM) was constructed for each contaminant for each full shift task at each site for each year of follow-up of the study (described in Benke et al., 2000). All individual exposures were assigned using the arithmetic mean for each job/task combination and those values were expressed in mg/m³. By combining the TEM with each worker’s job history, the cumulative exposure between interviews (years 3 mg/m3) was calculated for each constituent.

Data were analyzed with the Generalized Estimating Equations (GEE) method to account for the correlation among repeated measurements from each participant during follow up. Logistic models were used in analyses of health symptoms and BHR data and these models were adjusted for age, gender, age and gender interaction, tobacco smoking, smelter site, atopy and interaction between gender and smoking. Linear models were used in analyses of lung function and these models were also adjusted for age, gender, age and gender interaction, smoking, smelter site, height at entry interview and interaction between gender and smoking. Nearly all (98%) of the production, office (96%) and maintenance (95%) workers remained in the same group over the follow-up period. Cumulative exposures were described by tertile (but those data presented in the publisher’s appendix Table E2 were not available for review).

The highest prevalence and widest range of exposures were found for inhalable dust, but the empirical data were not presented. The 95thpercentiles for all airborne constituents considered in the Abramson et al. (2010) study were less than the current Australian Exposure Standards for an 8 h shift; only 1.1% of the cohort was exposed to total inhalable dust above the standard of 10 mg/m3. Asthma symptoms (wheeze and chest tightness) were associated with cumulative exposures to SO2 (p < 0.001 and p < 0.01, respectively), inhalable dust (p < 0.002 and p < 0.02) and coal tar pitch volatiles as the benzene soluble fraction (BSF) (p < 0.005 and p < 0.04). Fluoride (no details on the type of fluoride, e.g., gaseous or total) exposure was associated with wheeze (P < 0.04), but not with chest tightness.The authors reported that the association between wheeze and gaseous fluoride (OR 1.19, 95% CI 1.01 to 1.41 per tertile) was very similar to that for total fluoride (but those data were presented in publisher’s appendix Table E7 not available for review). When fluoride and inhalable dust were analyzed simultaneously for wheeze, only the coefficient for inhalable dust remained statistically significant (data from the publication referenced at appendix Table E4 were not available).Airflow limitation [the lower values of the forced expiratory ratio (FEV1/FVC)] was associated with higher cumulative exposures to coal tar pitch BSF (p < 0.03), fluoride (p < 0.04) and SO2 (p < 0.001). Longitudinal changes in pulmonary function (decline in FEV1 and FVC) were significantly associated with cumulative exposure to fluoride (p < 0.001 and p = 0.002, respectively), inhalable dust (p = 0.02 and p < 0.001, respectively) and SO2 (p = 0.001 and p = 0002, respectively).

Statistically significant associations were largely confined to male employees. The authors reported that the findings for gaseous F were similar to those for total F; however, data referenced at appendix Table E9 of the publication were not presented.The results suggested that the likelihood that bronchial hyper-responsiveness (BHR) increased significantly with cumulative exposure to coal tar pitch BSF (p = 0.03), fluoride (p = 0.03), inhalable dust (p = 0.001), SO2 (p = 0.009) and oil mist (p < 0.001).Bronchial hyper-responsiveness was associated with current tobacco smoking in females and atopy in both genders, but those data (referenced at publication appendix Table E11) were not presented. 

The strengths of the Abramson et al. (2010) study include the inception cohort design and robust assessment of workplace area exposures. The study is limited by possible misclassification of Al exposure including lack of personal sampling data (the results given in Benke et al., 2000; 2001). The data were expressed only as inhalable (total) dust and there was no differentiation between metallic Al and Al oxides. In addition, the fact data referenced in Tables E2-E11 were not available for review may limit interpretation of the results. The analyses were not adjusted for the use of personal protective equipment (PPE) and thus the reliability of the exposure data reported may be called into question. 

The Abramson et al. (2010) data establish relationships between cumulative occupational exposures to a number of airborne contaminants and occupational asthma among Al smelter workers. There was a significant association between chronic occupational SO2 exposure and wheeze and chest tightness, BHR reactions to methacholine challenge, reduced FEV1 and a longitudinal decline in lung function.A concentration-response relationship could be seen between fluoride exposure and those same outcomes, but the association was less evident. There was also a significant relationship between long-term cumulative exposure to inhalable dust and asthma-associated symptoms (wheeze and chest tightness), the longitudinal decline in pulmonary function (decline in FEV1 and FVC values) and increased BHR with the greater associations for wheeze and consecutive changes in FVC and BHR. Exposure to the BSF of coal tar pitch volatiles was also associated with increased asthma, airflow limitations and BHR, but coal tar pitch volatiles are not recognized as respiratory sensitizers. Oil mist exposure was also associated with increased BHR.Although many of the exposures were highly correlated, further statistical analyses suggested that of the known respiratory irritants, SO2 was more likely than fluoride to be responsible for many of the symptoms observed.In summary, chronic exposure to elevated levels of inhalable dust, SO2 and fluoride were the most important determinants associated with decrements in pulmonary function among these Al smelter employees. Again, no correlation to Al-levels was made and the study does not establish any relationship with Al-exposure.

Aluminum smelters - cardiopulmonary toxicity 

Friesen et al. (2010) studied acute and chronic polyaromatic hydrocarbon (PAH) exposure in relation to cardiopulmonary mortality in a cohort of 6,423 men and 603 women who worked for 3 or more years at an Al smelter in British Columbia, Canada. The authors linked data for the cohort to national mortality rates for the years 1957 to 1999 and examined exposure-response regarding the incidence of chronic respiratory diseases (COPD) and cerebrovascular disease. Work histories were abstracted from company records. Smoking status (ever smoking, never smoking, and unknown) was obtained through self-administered questionnaires sent to current workers, pensioners or to their survivors if the worker was deceased. Mortality of the cohort for select causes of death was compared with that of the whole of British Columbia using standardized mortality ratios (SMRs) adjusted for age, gender and time period. The development of a benzo(a)pyrene-based [B(a)P] quantitative job exposure matrix was described by Friesen et al. (2006). To create the [B(a)P] job-exposure matrix as a surrogate for total PAH exposures, statistical models were developed to derive annual arithmetic mean B(a)P levels for each operation and maintenance job in smelter potrooms that were based on personal exposure measurements collected from 1977 – 2000. This matrix accounted for different rates of exposure and declines in concentrations over time and potline. Exposure estimates for jobs without measurements were extrapolated from exposure estimates from the statistical models by adjusting for the amount of time worked in areas where ambient B(a)P levels had been determined.  Job- and time-period-specific B(a)P exposures levels were linked to each employee’s work history for calculation of cumulative and current B(a)P exposures. The models included smoking status and time-dependent covariates for years (5-year categories), time since first employed (years; continuous) and work status (employed at smelter: yes/no). For cerebrovascular disease and COPD, exposures were examined using cumulative B(a)P metrics (0-, 2-, 5-, and 10-year lags). The participants were workers who had a mean age of 32.4 years (range: 18–65), who were employed an average of 14.5 years (range: 3 – 45 years) and who contributed an average of 23.5 years (maximum 47) to study follow-up. Workers who died of ischemic heart disease (IHD) were more likely to have ever smoked than the average worker in the cohort (65% – 70% vs. 57%). The all-cause mortality SMR was less than that for the province’s population for both males (SMR = 0.87, 95% confidence interval (CI): 0.82 - 0.92) and females (SMR = 0.85, 95% CI: 0.63 - 1.11). Ischemic heart disease mortality (n = 281) was associated with cumulative historic B(a)P exposure (hazard ratio = 1.62, 95% confidence interval: 1.06, 2.46) in the highest category (> 66.7μg/m3-year). However, the higher hazard ratio for IHD found was for chronic B(a)P exposure and it was restricted to those who were in active employment (adjusted for smoking status and calendar year) - 2.39 (95% confidence interval: 0.95, 6.05) and who also had the highest cumulative B(a)P exposures (> 66.7μg/m3-year). The higher B(a)P exposures also had the widest confidence intervals. The stronger associations observed during employment suggest that cardiovascular effects may be reversible after termination of employment at the smelter even after adjusting for tobacco consumption. 

The Friesen et al. (2010) results suggest that cumulative workplace air PAH exposures in this Al smelter declined over time. One strength of the Friesen et al. (2010) study was its longitudinal design; however, the B(a)P exposures were not well characterized and exposure to Al and other constituents in smelter air was not taken into account. The authors elected to rely on mortality rates rather than morbidity for cardiopulmonary outcome and used semi-quantitative exposure estimates which may not reflect the actual PAH exposures. Failure to account for the host of factors known to influence cardiovascular health including diet, physical activity and the medical history of the participants are serious deficiencies. This study suggests there may be an association between heart disease and chronic PAH exposure in Al smelter workers. The study does not include an analysis of Al-exposure and related possible effects and is therefore not useful for the risk assessment of Aluminium.

 

Animal Studies

The high doses of particulates applied in animal studies tend to lead to overload-related effects (ATSDR, 2008; ILSI, 2000). Animal studies administering dust by intratracheal instillation (ITI) are not useful as sources of dose-descriptors for the inhalation route of exposure as some responses may result from the un-physiologic mode of administration. ITI studies can be useful, however, for screening and comparative ranking of particles for toxic effects. Several studies have shown interspecies differences in pulmonary reaction on exposure to aluminium metal and alumina (Engelbrecht et al., 1959; Gross et al., 1973; Christie et al., 1963).

Gross et al. (1973) did not observe development of alveolar proteinosis or thickening of alveolar walls in rats, hamsters or guinea pigs exposed to Al₂O₃ dust (66% < 1 μm). Pigott et al. (1981) reported no evidence of fibrosis in a repeated dose inhalation study that administered alumina fibres (Saffil) at levels between 2 and 3 mg/m³. The only pulmonary response observed was the occurrence of pigmented alveolar macrophages. 

Ess et al. (1993) investigated the subacute and chronic effects of short-term ITI administration (50 mg total dose, five 0.1 mL injections of suspension in sterile saline over a period of 2 weeks) of five smelter-grade and two laboratory-grade aluminas to Sprague-Dawley rats. These doses were sufficient to overload clearance mechanisms. All the dusts led to an inflammatory reaction in the alveoli evidenced through significantly elevated BALF total protein, LDH and sustained increases in PMN compared with the saline control. Only the laboratory grade aluminas showed signs of fibrosis (collagen) one year post-instillation, however.    

 

Adamcakova-Dodd et al. (2010) studied the pulmonary response following sub-acute inhalation exposure to aluminium nanowhiskers in mice. Aluminium nanowhiskers have been used in manufacturing processes as catalyst supports, flame retardants, adsorbents, or in ceramic, metal and plastic composite materials. Male mice (C57Bl/6J) were exposed to aluminium nanowhiskers for 4 h/day, 5 days/week for 2 or 4 weeks in the dynamic whole body inhalation exposure chamber. Control animals were exposed to a comparable sound level (80 dB) and laboratory air. The primary dimensions of these nanowhiskers were 2-4 nm x 2800 nm (Sigma-Aldrich) and the test nanomaterial [Al(OH)3:AlOOH] contained 35% Al.  The average concentration of aluminium nanowhiskers in the chamber was 3.3 ± 0.6 mg/m³ and median particle size diameter was 154.1 ± 1.6 nm. Both groups of mice were killed within 2 or 4 weeks of exposure at which time bronchoalveolar lavage (BAL) fluid was analyzed for differential and total cells, total protein, activity of lactate dehydrogenase (LDH) and cytokines. Total and differential white blood cells in the BAL fluid were counted. Lungs were processed for histopathology and pulmonary mechanics measurements were taken (flexiVent). The Al content in the lungs, heart, liver, spleen, kidney and brain was determined by ICP-OES. It was reported that the total number of cells as well as number of macrophages in BAL fluid was double that in mice exposed for 2 weeks and 6 times higher in mice exposed for 4 weeks, compared to air sham controls (p<0.01 and p<0.001, respectively). However, no neutrophilic inflammation in BAL fluid was found and the percentage of neutrophils was below 1% in all groups. No significant differences were found in total protein, activity of LDH, or cytokine levels (IL-6, IFN-γ, MIP-1α, TNF-α, and MIP-2) in BAL fluid between shams and Al-treated mice. In summary, sub-acute (2 or 4 weeks) inhalation exposures to Al whiskers increased the numbers of macrophages in BAL fluid. No other inflammatory or adverse responses were observed. Currently available information does not permit characterization of the nanowhiskers used in this study as particles or fibers.

This study provides new data on solubility and dissolution process of Al in biological fluids under different physiological conditions.However, the applicability of reported findings for manufactured Al2O3 nanomaterials to the bulk Al powders and dust is not clear.Only an abstract is available which limited interpretations of the study results. A Klimisch Score 4 (not assignable) was considered an appropriate for this study.

 

In vitro cytotoxicity studies

Aluminium metal reacts rapidly with air to form an aluminium oxide coat. Thus, exposure of tissues or cells to zero valence aluminium metal is unlikely by inhalation unless the aluminium powder is coated with a substance that acts as an effective barrier to oxidation in air but does not act as an effective barrier in the lung. The in-vitro studies by Wagner et al. (2007) and Braydich-Stolle et al. (2010) provide some evidence for a difference in cytotoxic effect between Al-NPs (2-3 nm oxide coat) compared with Al2O3-NPs. Al-NPs also showed an effect on macrophage phagocytosis of bacteria and particulates under the experimental conditions. The importance of this effect in-vivo in humans for larger particle sizes with thicker oxide coats is unclear. The utility of in-vitro studies for predicting the pulmonary toxicity profile in-vivo remains limited due to the dependence of biological effects, deposition, retention and inflammatory response on particle surface and physico-chemical characteristics (Sayes et al., 2007). 

 

Mechanism of Action

Overall, the results of the available in-vitro studies described earlier support the low cytotoxicity of poorly soluble aluminium oxide. Aluminium hydroxide and the closely related oxyhydroxide are similarly poorly soluble. These substances can be considered PSPs i.e. poorly soluble particulates of low cytotoxicity.

Summary

The current weight of evidence does not support a chemical-specific hazard on inhalation exposure to alumina (aluminium oxide, aluminium hydroxide) as experienced by the worker population. Gross et al. (1973) and Pauluhn (2009a) are considered the most adequate studies from which to obtain a dose descriptor to form the basis for a DNEL for repeated dose toxicity (inhalation, local effect) for these substances. The NOAEC from Gross et al. (1973), a subchronic study, for aluminium oxide (mean diameter 0.8 µm) is 75 mg/m³. The NOAEC from Pauluhn (2009a; sub-acute study; MMAD=1.7μm; agglomerated nanomaterials) for aluminium oxyhydroxide is 3 mg/m³ for a range of sensitive endpoints.

Considering aluminium oxide fume, the available information supports a low fibrogenicity (Stern and Pigott, 1983) and low cytotoxicity. 

 

Dermal

No animal studies are available in which the repeated exposure toxicity of aluminium has been investigated.

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008 classification criteria (CLP) for repeated dose toxicity, no classification is required.