Rapal 01, Paval, Serox, Argalum

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

The investigation was reported in a scientific paper without specifying whether GLP conditions were applied.

Type of assay:

mammalian erythrocyte micronucleus test

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

Molecular formula:

Al2O3

IUPAC Name:

aluminium oxide

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Institute of Nutrition, Hyderabad, India
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 90 - 100 g
- Housing: Rats were kept in polypropylene cages under controlled conditions
- Diet: Animals received a 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%), ad libitum.
- Water: Animals received a standard laboratory water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 60 ± 10
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DDW-Tween 80 (1%) mixture
- Injected Volume: Unclear
- Injected concentration: Unclear as volume was not provided.
- Amount of vehicle (if gavage or dermal): The amount of test substance injected was 47.5 mg, 95 mg and 190 mg Al2O3, equivalent to 25 mg, 50 mg and 100 mg of Al (calculated from the numbers provided in the article).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Al2O3 particles were 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:

Genotoxicity Assays
Exposure Duration: Single exposure
Schedule/Frequency of Administrations: Single, acute

Frequency of treatment:

Not applicable.

Post exposure period:

MN assay: 30 h, 48 h

Mitotic Index (MI): 18 h, 24 h

Al Level Study:
Urine and faeces: 48 h after dosing
Blood and tissues: 14 days after dosing

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Al2O3; corresponding to an actual amounts of 47.5 mg Al2O3 assuming a body weight of 95 g or 25 mg Al

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Al2O3; corresponding to an actual amounts of 95 mg Al2O3 assuming a body weight of 95 g or 50 mg Al

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

Al2O3; corresponding to an actual amounts of 190 mg Al2O3 assuming a body weight of 95 g or 100 mg 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(s): intraperitoneal
Dose/concentration: 40 mg/kg bw, volume: 0.01mL/g bw

Examinations

Tissues and cell types examined:

Rat bone marrow.

Details of tissue and slide preparation:

MN assay:
The authors stated that the measurements were made in accordance with OECD TG 474. A minimum of 4 slides were made for each animal for assessment of MN frequency and stained with Giemsa. Slides were coded. A total of 2000 PCEs (polychromatic erythrocytes) from all 4 slides were scored for MN.

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:

The criteria used for a positive response were not provided explicitly but the guidelines were cited.

OECD TG 474: reports that there are several criteria for determining a positive result e.g. dose-related increase or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. “Biological relevance of the results should be considered first.”

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

Sex:

female

Genotoxicity:

negative

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.

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 18 h:
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 24 h:
Negative control: 3.25 ± 0.10
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

MN Assay:

OECD TG 474: Principal endpoint = Frequency of micronucleated immature (polychromatic) erythrocytes

 

The results of the ANOVA omnibus test for a difference between groups was not provided.

None of the treated groups had significantly lower %PCEs compared with the control group.

 

Negative control (1% Tween 80)

MN-PCEs/2000 PCEs, mean ±sd

30 h: 2.5 ± 0.70

48 h: 1.8 ± 0.75

Al₂O₃- (50 - 200 μm)

30 h and 48 h: The frequency of MN-PCEs increased with dose but the pair-wise comparisons with the negative control were not significant. The standard deviation of the results (based on 5 animals per dose) showed an increase with dose.

MN-frequencies at 30 h and 48 h were similar.

30 h:

25 mg Al: 1.9±0.73, ns

50 mg Al: 3.3±1.16, ns

100 mg Al: 5.9±1.71, ns

48 h:

25 mg Al: 2.0±0.64, ns

50 mg Al: 4.2±1.07, ns

100 mg Al: 6.6±1.68, ns

Applicant's summary and conclusion

Conclusions:

The genotoxicity results for 50 to 200 μm diameter particles (Al2O3-bulk) were not significantly different from those of the vehicle control.

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.

Executive summary:

Balasubramanyam et al. (2009a) determined the frequency of micronuclei in polychromatic (immature) erythrocytes (MN-PCEs) in bone marrow according to OECD Test Guideline 474 (1997). The percentage of PCEs was not significantly different from the vehicle control (1% Tween 80 in DDW) in any treated group indicating that cell death was not occurring as a result of treatment. The results were negative for the 50 to 200 µm sized Al2O3-bulk particles.

Balasubramanyam et al. (2009a) also reported measurements of levels of Al in the urine and faeces sampled 48 hours after dosing and in tissues in samples taken 14 days after dosing. The table showing the tissue analysis results was almost identical to that in Balasubramanyam et al. (2009b) with the exception that the tissue values were reported as “Al content” in contrast to “Al2O3 content” in Balasubramanyam et al. (2009b). Contact with the author clarified that the units used in the table for tissue doses were μg Al2O3/g wet tissue. 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 in levels of Al in tissues and organs with increasing dose. The units used in the article for levels in urine (µg/mL) indicate Al concentrations in the urine of the control group to be 3-orders of magnitude higher than the normal range in humans (1-2 µg/L) (Krewski et al., 2007). The reliability of the aluminium measurements in tissues and urine is limited by the inconsistencies and lack of clarity in reporting. A Klimisch Score of 3 was assigned to the Al tissue measurements.