Aluminum iron oxide (Al2FeO4)

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-12-09 - 2010-04-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Spinel (Mg(AlO2)2)

EC Number:

215-105-9

EC Name:

Spinel (Mg(AlO2)2)

Cas Number:

1302-67-6

Molecular formula:

AlO2.1/2Mg

IUPAC Name:

magnesium dialuminium oxide

Test animals

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

Hygiene Optimal hygienic conditions.
Room No. EI1-10.
Room temperature Average of 19.8 °C (see Table 19).
Relative humidity Average of 59.2 % (see Table 19).
Air exchange 12 per hour.
Light Artificial light from 6 a.m. to 6 p.m.
Cages 4-fold caging (animals of one group and one sex per cage). Makrolon cages type IV (33 cm x 55 cm area, 20 cm height).
Bedding material Aspen wood chips (ABEDD Dominik Mayr KEG, A-8580 Köflach), autoclaved. Bedding material was changed weekly. Acceptable contaminants are identical with the limits for contaminants in the feed, see below.
Feed Ssniff R/M-H maintenance diet for rats and mice (item V1534-3) ad libitum, supplied by Ssniff Spezialdiäten GmbH, 59494 Soest, Germany. Exception: Feed was withdrawn on days prior to blood sampling at 5:00 p.m., only from the animals, where blood was to be taken, and was re-offered immediately after the blood sampling. Random samples of the feed are analysed for contaminants by the supplier. One sample is analysed also for contaminants in addition by an independent external laboratory. The limits of tolerance are derived from the "Deutsche Futtermittelverordnung" (German feed regulation).
Water Tap water, acidified with HCl to pH 3, from an automatic watering system, ad libitum. Random samples of the water are analysed by the "AGES", 1226 Vienna, Austria, to check, if the water fulfils the requirements for drinking water for humans (exception: the pH).
Environmental enrichment Nibbling wood bricks (sized 10 cm x 2 cm x 2 cm), same material and source as the bedding material, were offered freshly once a week.
Identification Tattoo in the right pinna.
Acclimatization At least 5 days.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The size of the dust particles was analysed with a cascade impactor (Berner-Impaktor Type LPI4/0,06/2 from Hauke KG, Gmunden, Austria). It contains nine steps with cut-off- diameters from 0.06 µm to 16 µm. The cut-off diameters were obtained from the manufacturer.
The mass median aerodynamic diameter of the dust particles was 4.0, 4.5 and 5.5 µm for the low, mid and high concentration. This is somewhat higher than the recommended range of 1 to 4 µm but as the shape of the test substance powder particles is a characteristic property of this product, the powder was not ground to decrease the size. As the high concentration was also the highest technically feasible concentration also no separator for larger particles was used as this would decrease also the concentation of smaller particles to some extent.

Details on inhalation exposure:

The test substance powder was pressed to a powder cake. Portions of 25 g each were filled into a steel cylinder with 46 mm inner diameter and pressed with a force of 10 tons. The whole powder cake consisted of 6 such portions, i.e. 150 g powder. These steel cylinders with the powder cake were used to for the filling of the dust generator.
The animals were exposed to dusts of the test substance 5 days per week for 4 weeks. The desired dust concentrations were produced with dust generators according to Budiman, using pressed dust cakes.
The route was defined by the sponsor. The administration per inhalation is a common route in toxicity testing of chemicals which consist of small particles and may produce dusts during handling.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dust concentration in the breathing zone was determined gravimetrically. An accurately measured volume of air from the inhalation devices was sucked through a pre-weighed filter with cotton wool. The filters were dried before and after the sampling by pressing dry air through them. From the weight difference and the volume the actual dust concentration was calculated.

Duration of treatment / exposure:

6 hours per day, fife days per week, for 4 consecutive weeks. The satellite groups were kept for additional 2 weeks without further dosing.

Frequency of treatment:

five days per week, for 4 consecutive weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.3 mg/L
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.95 mg/L
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
2.77 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

Dose Range Finding: 12 males and 12 females
Main Study: 32 males and 32 females. 8 males and 8 females per group.
Females were nulliparous and non-pregnant.

Control animals:

yes

Details on study design:

Route of test substance administration: Per inhalation as a dust.
Dosing regimen: 6 hours per day, fife days per week, for 4 consecutive weeks. The satellite groups were kept for additional 2 weeks without further dosing.
Inhalation system: Head Nose Only Exposure Unit for the Inhalation of Aerosols.
Test system (animals): Rats, Fischer, F344/DuCrl. 5 males and 5 females per main group, and additionally 3 males and 3 females per lavage group.
Groups, doses:
• K (negative control group) air only,
• KS (negative control satellite group) air only,
• A (low dose group) 0.30 mg / L,
• B (mid dose group) 0.95 mg / L,
• C (high dose group) 2.77 mg / L,
• CS (high dose satellite group) 2.77 mg / L,
The doses are derived from and based on the results of a dose range finding study. For a survey thereof, see below.
Investigations:
• Analyses of the dust concentrations:
Three times per day.
• Analyses of the dust particle size:
Once per week.
• Animal observations:
All animals, before and after the exposure, on days without exposure once a day.
• Body weights:
All animals, twice per week.
• Feed consumption:
All animals, for weekly periods.
• Haematology:
5 males and females of groups K, A, B and C on Day 29 and 5 males and females of groups KS and CS on Day 43.
• Clinical biochemistry:
Times and animals like Haematology.
• Necropsy with gross pathological examination:
5 males and females of groups K, A, B and C on Day 29 and 5 males and females of groups KS and CS on Day 43.
• Organ weight determination:
Selected organs in 5 males and 5 females at necropsy.
• Histopathological examination:
Selected organs or tissues in 5 males and 5 females of groups K and C. Organs and tissues with suspected test substance related alterations in all groups.
• Bronchoalveolar lavage (BAL):
3 males and females per group. Determination of total protein and LDH activity. Counting of viable and dead cells. Microscopic examination of a sediment smear after centrifugation for cell differentation.
• Determination of aluminium and magnesium in the serum:
Determined in 3 males and 3 females per group at necropsy.

Examinations

Observations and examinations performed and frequency:

Daily observations in life
All animals were carefully observed for general signs and the health status once a day.

Body weights
The individual body weights were determined on Day 1 , Day 3 and Day 8 of the Dose Range Finding Study.

Feed consumption
Determined for all animals for the entire administration period.

Necropsy
The animals were killed by exsanguination from an axillar artery in deep anaesthesia (Ketamine i.m.) and subjected to a gross pathological examination on Day 8.

Investigations in the Main Study
Observations in life
All animals were carefully observed for general signs and the health status before, during and after the exposure; on days without exposure once a day.

Body weights
The individual body weights were determined twice per week for all animals.

Feed consumption
Determined per cage in weekly intervals in all animals.

Haematology
Blood samples were taken from the retrobulbar vein plexus of the left eyes in slight ether anaesthesia in the morning after overnight fasting.
Feed was offered again immediately after the blood sampling.
Blood was taken on Day 29 from the first 5 males and females of groups K, A, B and C; and of the first 5 males and females of groups KS and CS on Day 43.
Parameters determined (abbreviations, when commonly used):
• Red blood cell count (RBC)
• Haemoglobin concentration (Hb)
• Haematocrit (Hct)
• Mean corpuscular haemoglobin (MCH)
• Mean corpuscular haemoglobin concentration (MCHC)
• Mean cell volume (MCV)
• Reticulocytes count (% of erythrocytes)
• White blood cell count (WBC)
• Platelet count
• Differential white blood cell count (% of the different cell species)
• Prothrombin time (Quick) as indicator of blood clotting capacity
Haematological examination of the blood samples, with the exception of the differential white blood cell count, was performed at the "Ambulatorium für med. u. chem. Labordiagnostik", A-7000 Eisenstadt, using Coulter STKS – Instrumentation Laboratory.
The differential white blood cell count (by microscopic examination) and the prothrombin time (by test kits by Roche) were determined in house.

Clinical biochemistry
Blood samples were taken from the same animals and at the same time as for haematology. Plasma (with Li-heparin) was obtained by centrifugation of the blood samples.
Parameters determined (abbreviations, when commonly used):
• Alanin aminotransferase (ALT, GPT)
• Albumin
• Alkaline phosphatase (AP)
• Aspartate aminotransferase (AST, GOT)
• Bilirubin
• Calcium
• Chloride
• Cholesterol
• Creatinine
• Gamma glutamyl transferase (GT, GGT)
• Globulin (calculated)
• Glucose
• Phosphorus
• Potassium (K+)
• Sodium (Na+)
• Total protein
• Triglycerides
• Urea
Clinical-biochemical examination of the plasma samples was performed at the "Ambulatorium für med.chem. Labordiagnostik", A-7000 Eisenstadt, using a Roche Hitachi 917 for the chemical parameters and an ion-selective electrode for electrolyte determinations.

Sacrifice and pathology:

Necropsy
The animals are killed by exsanguination from an axillar artery in deep anaesthesia (Thiopental, i.p. or Ketamine i.m.) and subjected to a necropsy including a gross pathological examination immediately after death on

• Day 29 (first 5 animals of each sex of groups K, A, B and C)
• Day 43 (first 5 animals of each sex of groups KS and CS)
The following organs/tissues (if appropriate) were fixed in 4 % buffered formaldehyde, with the exception of the eyes (fixed in Davidson's fixative) and the testes (fixed in Bouin's solution, punctured on both poles with a needle before immersion in the fixative):

gross lesions, tissue masses or tumours ovaries
adrenal glands seminal vesicles
brain (including cerebrum, cerebellum and pons) spinal cord (cervical, thoracal, lumbar)
eyes * spleen
heart sternum with bone marrow
kidneys stomach
larynx (3 levels, including the base of the epiglottis) testes
liver thymus
lungs (all lobes at one level, including main bronchi) thyroid glands
lymph nodes (hilar region of the lungs) trachea (longitudinal section through the carna and 1 transverse section)
nasopharyngeal tissues (4 levels) urinary bladder *
oesophagus uterus
olfactory bulb *
*) Not included in routine histopathology.

Organ weights
Wet weights of the following organs are determined from all above mentioned animals in the course of the necropsy (fresh weights):

• adrenal glands (both together)
• brain
• heart
• kidneys (both together)
• liver
• lungs
• spleen
• testes (both together)
• thymus

Relative organ weights were calculated by relating the absolute organ weights to the last determined individual body weight and to the brain weight.

Histopathology
Groups K and C:
Histopathological examination was performed in the first 5 males and females of all fixed organs and tissues listed above (see 2.8.2.6), except those, labelled with a "*"..
Groups KS, A, B and CS:
All organs and tissues with treatment related changes in group C were examined.
For a list of organs and tissues examined histopathologically or of missing organs/tissues see Table 28.
The tissue trimming was performed according to "Bahnemann et al.: RITA - Registry of Industrial Toxicology Animal Data - Guides for Organ Sampling and Trimming Procedures in Rats"; Exp.Toxicol.Pathol. 47 (1995), p 247 ff. with the following exceptions:
Not all possible sections, as given in the literature, were actually prepared. One section per organ (in paired organs one of each) was made with the following exceptions:
• Brain (3 sections, one at the optic chiasma, the second at the caudal border of the mammillary body, just posterior to the attachment of the pituitary and the third about 2 mm caudal to the transverse fibres of the pons). Representative regions of the brain, especially cerebrum, cerebellum and pons are included in these sections.
• Spinal cord (three sections, a cervical, a thoracal and a lumbar).
• Liver (two sections).
The trimmed samples of organs or tissues, as described above, were embedded in paraffin. Sections of about 5 m were stained with haematoxylin and eosin (H&E). Evaluation of slides was performed using a light microscope Leica-DMRB.
To describe the severity of lesions, the following grades were applied, if appropriate:
minimal (1), mild (2), moderate (3), marked (4), severe (5).
The term "focal" together with a higher degree of severity also stands for "multifocal".

Other examinations:

Bronchoalveolar lavage (BAL):
Animals were killed by inhalation of 80 % CO2 plus 20 % air on
• Day 29 (last 3 animals of each sex of groups K, A, B and C)
• Day 43 (last 3 animals of each sex of groups KS and CS)
The lavage was performed by instillation of 5 mL isotonic saline into the trachea and reharvesting the fluid.

Examinations performed in the lavage fluid:
• Determination of total protein (according to Lowry)
• Lactate dehydrogenase (LDH) activity. Performed at the "Ambulatorium für med.chem. Labordiagnostik", A-7000 Eisenstadt, using a Roche Hitachi 917
• Counting of viable and dead cells in a sediment after centrifugation and staining with trypan blue.
• Microscopic examination of a sediment smear after centrifugation (Giemsa stain) for cell differentation.

Determination of aluminium and magnesium in serum:
Before the animals were killed for bronchoalveolar lavage, blood samples were taken from the retrobulbar vein plexus of the left eyes in slight ether anaesthesia. Serum was produced by centrifugation after clotting. The serum samples were frozen and handed over to the analytical laboratory. Prior to analyses the samples were defrosted and an aliquot of about 0,5 g of serum of each sample was digested with nitric acid. The resulting solutions were analysed using inductively coupled plasma mass spectrometry (ICP-MS).
Test system and measuring conditions:
Test system: ICP-MS Model ELAN 6100 with autosampler AS90 from Perkin Elmer Sciex
Software: Perkin Elmer Sciex Elan version 2.3.2
Nebulizer: Cross flow
Plasma power: 1100 W
Sample flow: 1.2 mL / min (continuous)
Nebulizer gas flow: 0.84 L Ar / min
Plasma gas flow: 15 L Ar / min
Calibration model: Linear regression
Number of replicates: 6

Bias control
Random numbers were used for
• allocation of the individual animals to their groups,
• sequence of animals for blood sampling,
• sequence of haematological and clinical biochemistry analyses,
• sequence of animals for necropsy,
• sequence of animals for histopathological examination (group K and C).
The sequence of groups for the starting of the exposure to the test substance dust was changed daily. The position of the racks within the animal room was changed twice a month to avoid systemic influences by unknown factors.

Statistics:

Statistical methods
statistical method used for
Analysis of variance followed by the Scheffé-test all data with means and standard deviations determined, comparison of more than two groups
t-test all data with means and standard deviations determined, for comparison of two groups only
H-test of Kruskal and Wallis followed by the test of Nemenyi counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled
Chi2-test counted events
Fisher's exact test counted events, if the Chi2-Test was not applicable

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gains were generally somewhat lower in the high concentration group. There were no statistically significant changes in the low and mid dosed group. The body weight in the low concentration group was usually even slightly higher then in th

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

All observed changes are too small to give an indication for a toxic effect. They may be random events or side effects of the adaptive reaction of the lungs.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The weight of the lungs was clearly increased in all dosed groups, gaining statistically significance in the mid and high dosed animals. These weight changes did not reveal during the recovery period. This is probably caused by the adaptive reaction.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Except for the alterations associated with the airways, no test substance related findings were made histopathologically. All other findings are interpreted as spontaneous ones, inconspicuous in type and incidence.

Effect levels

Dose descriptor:

NOAEL

Basis for effect level:

other: no NOAEL identified

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Haematology

parameter (sex)	low dose 0.30mg/L (% of the negative controls)	mid dose 0.95mg/L (% of the negative controls)	high dose 2.77mg/L (% of the negative controls)	high dose after recovery 2.77mg/L (% of the negative controls)
lymphocytes, % of leukocytes(males)	91	84	82	83
neutrophils, % of leukocytes(males)	137	162	164	150
neutrophils, cells/nL(males)	135	154	171	164

The changes in the lymphocytes/neutrophles ratio may be caused by adaptive/inflammatory reactions of the lungs to the test substance dust. The changes in females were less pronounced and did not reach statistical significance, but they were in the same direction than in males.

Clinical biochemistry

parameter (sex)	low dose 0.30mg/L (% of the negative controls)	mid dose 0.95mg/L (% of the negative controls)	high dose 2.77mg/L (% of the negative controls)	high dose after recovery 2.77mg/L (% of the negative controls)
potassium(males)	106	110	105	95
triglycerides(males)	78	79	67	93
glucose(males)	84	82	78	95
calcium(females)	100	100	101	102
alkaline phosphatase(females)	103	100	107	117
aspartate aminotransferase(females)	111	105	116	115
alanin aminotransferase(females)	101	105	107	127

All observed changes are too small to give an indication for a toxic effect. They may be random events or side effects of the adaptive reaction of the lungs.

Organ weights

parameter (sex)	low dose 0.30mg/L (% of the negative controls)	mid dose 0.95mg/L (% of the negative controls)	high dose 2.77mg/L (% of the negative controls)	high dose after recovery 2.77mg/L (% of the negative controls)
lung(males, absolute weight)	134	155	160	154
lung(males, organ weight/body weight ratio)	130	153	167	151
lung(males, organ weight/brain weight ratio)	135	155	162	153
lung(females, absolute weight)	117	141	150	140
lung(females, organ weight/body weight ratio)	117	142	151	142
lung(females, organ weight/brain weight ratio)	122	143	152	137

Histopathology:

The test substance dust caused several effects in the nasal cavity, the larynx, the bronchial lymph nodes and the in the lungs. Most effects were adaptive responses to the deposits of the test substance and the resulting irritation (goblet cell proliferation in the nasal cavity, focal metaplasia at the basis of the epiglottis, phagocytosis). There were also findings of developing fibrosis and a chronic inflammatory response, though always in a low grade of severity and also only in some animals. These findings may be caused by the overloading of the lungs with test substance dust.

Bronchoalveolar lavage

Lactate dehydrogenase (LDH) and protein concentration was increased in all dosed groups of both sexes. The number of cells in the lavage fluid was significantly increased and the relation of lymphocytes and neutrophiles was changed in all dosed groups of both sexes. The fraction of non viable cells increased only very slightly with the dose and did not reach statistically significance. The test substance dust was obviously inert against the cells. All these changes can be explained as a response of the lungs to the deposit of the solid test substance particles and the irritation caused by them.

Aluminium and magnesium in serum

There was no difference in the magnesium concentration in the dosed groups. All aluminium concentrations (with one exception which is probably an outlier) were under or near the detection limit of 0.5 mg/kg. There was therefore no indication that the test substance releases magnesium or aluminium.

Applicant's summary and conclusion

Conclusions:

The test substance dust at concentrations of 0.30 to 2.77 mg/L caused effects at several sites of the respiratory system. In the upper airways, i.e. the nasal cavity and the larynx, there were indications for a minimal local irritation. The alterations are given only limited toxicological relevance.
In the lungs, large amounts of the unchanged test material were stored in the alveoli or within phagocytes. Some material was also found in the regional lymph nodes. Except for sheer storage, early stages of fibrosis and granulomatous foci were repeatedly noted in the lungs. The low grade of the latter alterations corresponds with the relative short time of test substance exposure. Those changes were not very pronounced in the present study, but might grow with longer exposure time. Due to the high amount of test substance deposits in the lungs, these changes may be explained by simply overloading the tissue and do not necessarily imply an intrinsic toxicity of the test material. An intrinsic toxicity is unlikely as the test material is insoluble and the shape of the particles is not fibrous.
The test substance did not dissolve in the organism of the animals. No increased amounts of magnesium or aluminium were found in the sera of the rats.
There was no substantially sex difference in the response to the test substance.
The effects noted were at most mild in severity, never became life threatening, but persisted partly until the end of the recovery period.
The No-observed-effect-level (NOEL) of "Spinel" dust was smaller than 0.30 mg/L, as effects were also seen in the low concentration group. There was however no life threatening effect present, not even in the high concentration group. But early stages of fibrosis and granulomatous foci were repeatedly noted in the lungs which might grow with longer exposure time to such high dust concentrations.

Due to the extremely high concentration of the dust (even the lowest concentration of 0.30 mg/L is equivalent to 300 mg/m³!) obviously the lungs were overloaded. This is very much in agreement with the observations that no concentration effect relationship could be determined and that the lungs were stuffed with the unchanged test substances.
Any substance specific toxicity was not observed, in particular it could be shown that despite this extremely high exposure cell viability of lavaged lung cells was not significantly effected.
Also further supporting evidence is available that the test substance is practically biologically inert due to its very low dissolution and hence bioavailability, i.e. no cell toxicity in in vitro assays, no sensitizing potency.
Therefore it is concluded that the test substance Spinel as a dust fulfils the definition for inert (nuisance) dust for which airborne concentrations of 10 mg (for chronic) and of 20 mg (for acute exposure respectively) for respirable dust and of 3 mg (for chronic) and of 6 mg (for acute exposure respectively) for alveolar dust have been defined.

Some effects from the inhalation of the test substance (at 300 mg/m³!) could only be observed at approximately at minimum two orders of magnitude the concentration of the inert dust threshold (of 3 mg/m³) for alveolar dust.

Executive summary:

Dust from Spinel was tested at very high concentrations in a subacute inhalation toxicity assay for its potency to adversely react with lung tissue. The observed effects clearly follow a pattern of an overload phenomenon, since neither a concentration effect relationship nor any significant decrease of cell viability could be observed.

Since Spinel also does not dissolve and is therefore not bioavailable and is practically biologically inert in variousin vitrotest systems dust from Spinel fulfils the definition of inert (nuisance) dust.

It can be therefore assumed that Spinel as a dust fulfils the definition for inert (nuisance) dust for which airborne concentrations of 10 mg (for chronic) and of 20 mg (for acute exposure respectively) for respirable dust and of 3 mg (for chronic) and of 6 mg (for acute exposure respectively) for alveolar dust have been defined.