Aluminium triisopropanolate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Evaluation of genotoxic effects of oral exposure to aluminum oxide nanomaterials in rat bone marrow

Author:

Balasubramanyam A, Sailaja N, Mahboob M, Rahman MF, Misra S, Hussain SM, Grover P.

Year:

2009

Bibliographic source:

Mutation Research 676 (1-2): 41-47.

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium oxide

EC Number:

215-691-6

EC Name:

Aluminium oxide

Cas Number:

1344-28-1

IUPAC Name:

aluminium oxide

Specific details on test material used for the study:

purity > 90%
particle size: 50-200 um

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: W NIN (National Institute of Nutrition, Hyderabad, India).
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 90-100 g
- Diet: ad libitum. The feed was described as standard laboratory feed (wheat flour 2.5%; roasted Bengal gram flour 60%; skimmed milk powder 5%; casein 4%; refined ground oil 4%; salt mixture 4%; vitamin mixture 0.5%).
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12-h light/dark cycles

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DDW-Tween 80 (1%) mixture
- Volume dosed: No data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Al2O3 was suspended in 1% Tween 80 (a surfactant that enhances uptake), dispersed by ultrasonic vibration for 10 minutes and “mixed thoroughly” prior to use.

Duration of treatment / exposure:

14 days

Frequency of treatment:

acute exposure (expected to be daily)

Post exposure period:

CA assay: 18h, 24 h

Mitotic Index (MI): 18h, 24h

Al Level Study:
Urine and faeces: 48 h after (first?) dosing
Blood and tissues (liver, spleen, heart, kidneys and brain): on day 14 at sacrifice

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

as Al2O3; 265 mg/kg bw as Al

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

as Al2O3; 529 mg/kg bw as Al

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

as Al2O3; 1069 mg/kg bw as Al

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Substance: Cyclophosphamide
Justification: Known mutagen on list of acceptable positive control substances in the relevant OECD TGs.
Route of administration: intraperitoneal
Dose/concentration: 40 mg/kg bw, volume = 0.01 mL/g bw

Examinations

Tissues and cell types examined:

Rat bone marrow.

Details of tissue and slide preparation:

CA Assay:
The authors stated that the measurements were made in accordance with OECD TG#475. 500 well-spread metaphases (100 per animal) were analyzed for each treatment (dose/time). Aneuploidy, polyploidy, gaps, breaks, minutes, acentric fragments and reciprocal translocations were counted.

Al-Levels:
0.1 - 0.3 g of fresh tissue were predigested in ultrapure nitric acid overnight, heated to 80 ºC for 10 h and then 130 - 150 ºC for 30 minutes. The samples were then heated (temperature not provided) for an additional 4 hours in the presence of 0.5 mL 70% perchloric acid and evaporated to dryness. Solutions were then made up to 5 mL with deionized water, filtered and the Al concentration was determined using ICP-MS with rhodium at 20 ng/mL as an internal standard.

Cytotoxicity (Mitotic Index)
The MI was determined on 1000 cells or more from randomly selected slides that were coded prior to scoring.

Evaluation criteria:

According to guideline.

Statistics:

The methods used were included in the article but were not well-described. The LSD-test mentioned in the article is more appropriate for particular planned comparisons and not for comparing several pairs of means. Testing for homogeneity of variances was also not mentioned. The results show some evidence of an increase in the magnitude of the standard deviation with dose for some endpoints.

Results and discussion

Test results

Key result

Sex:

female

Genotoxicity:

negative

Remarks:

CA Assay: Al2O3 - (50-200 μm)

Toxicity:

no effects

Remarks:

The authors briefly mention that no mortality nor toxic symptoms were observed at any dose level in the range-finding study (OECD TG #420) nor in the 5 rats at the highest dose level in the main study that was reported in the article.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MI: There was no significant reduction in MI at either sampling time for any of the dose groups compared with the vehicle control.

At 18h:
Negative control: 2.97(±0.12)
Treated groups: The MI ranged from 91 to 110% of the value in the negative control.
Positive control: 75% of negative control
At 24h:
Negative control: 3.25±0.10 (mean, sd)
Treated groups: The MI ranged from 82 to 90% of the value in the negative control.
Positive control: 80% of negative control

General toxicity: The authors briefly mention that no mortality nor toxic symptoms were observed at any dose level in the range-finding study (OECD TG #420) nor in the 5 rats at the highest dose level in the main study that was reported in the article.

Any other information on results incl. tables

CA assay:

Total aberrations (excluding gaps)

 

Negative control (1% Tween 80), mean (±sd)

18h: 0.6(±0.32)

24h: 0.5(±

0.42)

 

Al₂O₃- (50-200 μm)

18h:

25 mg Al:0.6±0.22 - ns

50 mg Al: 2.2±0.79 - ns

100 mg Al: 4.3±1.01 - ns

24h:

25 mg Al:0.5±0.31 - ns

50 mg Al: 1.2±0.51 - ns

100 mg Al: 5.5±0.31 - ns

- No polyploidy or reciprocal translocations observed in the Al-treated groups;

- Aneuploidy: clear dose response (not statistically tested for trend) observed for the 30 nm and 40 nm groups with significantly higher numbers in the highest dose group (p<0.001). Al-levels: “Al-content”μgAl₂O₃/g wet tissue:

Al Levels

Control (units±sd)

Whole blood   3±1 μg/g

Liver              6±2μg/g

Spleen            2±1μg/g

Heart             6±3 μg/g

Kidneys          9±4 μg/g

Brain              2±3 μg/g

Urine (48 hours post-dosing) 5±2 μg/mL

Faeces (48 hours post-dosing) 1±1 mg/g

 

Al₂O₃- (50-200 μm)”bulk”

The levels in tissues show an increase with dose that does not reach statistical significance. A significant (p<0.01) increase in the Al₂O₃content in faeces relative to the control was observed at 2000 mg/kg bw. The amounts of Al in tissues of the nanomaterial dosed animals were much higher than those found in animals dosed with the bulk material.

A particle size dependence of gastrointestinal absorption was apparent with lower levels in the tissues reported for the larger 50 to 200 μm diameter particles (Al2O3-bulk).  The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls. The reported levels of Al in the urine of the control group were three orders of magnitude greater than “normal” levels in humans.

Applicant's summary and conclusion

Conclusions:

No significant increase in the number of aberrations. Therefore it can be concluded that Al2O3 does not exhibity clastogenic effects under the conditions of the test

Executive summary:

Balasubramanyam et al. (2009a) administered suspensions of aluminium oxide by oral gavage to female albino Wistar rats (5 animals per group). Concentrations of 500, 1000 and 2000 mg Al2O3/kg bw in 1% Tween 80/doubly-distilled water (DDW) were administered to the rats. These concentrations are equivalent to 265, 529 and 1059 mg Al/kg bw. Three size fractions of aluminium oxide particles were examined: Al2O3 (30 nm; transmission electron microscopy (TEM) determined diameter, mean±sd - 39.85±31.33 nm), Al2O3 (40 nm; TEM diameter, mean±sd – 7.33±36.13 nm), and Al2O3-bulk (diameter 50 to 200μm). A negative control group was treated orally with the Tween 80/DDW vehicle.A positive control group received a single intraperitoneal dose of 40 mg/kg bw of cyclophosphamide. The assessment of CA in bone marrow cells was conducted in accordance with OECD Test Guideline #475 with the analysis of 500 well-spread metaphases (100 per animal) for each treatment 18 and 24 hours after the last dosing. Aneuploidy, polyploidy, gaps, breaks, minutes, acentric fragments and reciprocal translocations were counted. The mitotic index was determined on 1000 cells at both sampling times, slides were selected randomly and coded to blind analysts. Dose levels were determined using an acute oral toxicity study conducted in accordance with OECD Test Guideline #420. Organ tissue was analyzed for aluminium content by ICP-MS (inductively coupled mass spectrometry). The LSD-test mentioned in the article is more appropriate for particular planned comparisons and not for comparing several pairs of means. There was no indication of an effect of treatment on the mitotic index. 

Eighteen hours after the final dosing, the mean±sd total aberrations for the control, 265, 529 and 1058 mg Al/kg bw/day Al2O3-bulk groups were 0.6±0.3, 0.6±0.3, 2.2±0.8, and 4.3±1.0, respectively. Statistical testing reported in the article indicated no significant differences for any treatment level of this group compared with the control group. The reported measurements of levels of Al in the urine and faeces sampled 48 hours after dosing and in tissues in samples taken on day 14 were reported as μg Al2O3/g wet tissue (clarified after contact with the author). Aluminium levels were elevated in all tissues in a dose-response manner with either of the nano-sized particulates. A particle size dependence of gastrointestinal absorption was apparent with lower levels of aluminium in the tissues reported for the larger 50 to 200 μm diameter particles (Al2O3-bulk). The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls. The measurements do, however, show consistent increases inlevels of Al in tissues and organs with increasing dose.