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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from supporting substance, but study conducted according to OECD guideline 474 and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Purity 99% as stated by sponsor

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male
Details on test animals and environmental conditions:
Test animals were obtained from Charles River (UK) Ltd, Margate, UK. Animals were housed in air-conditioned room (min. 15 air changes/hour). Temperature range: 19-25°C; relative humidity range: 40-70%.
Light/Dark cycle: 12/12 hours.
The animals were housed in roups of up to 6 of the same sex.
Diet, water, bedding: ad libitum access to SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham), mains water provided ad libitum via water bottles, bedding provided on a weekly basis to each cage (European softwood bedding, Datesand Ltd, Manchester).

Pre-experimental procedures:
- Acclimatisation and health procedures: clinical inspection on arrival, acclimatisation phase of at least 5 days, veterinary inspection before start of dosing.
- Allocation to treatment group: Range-finder animals were allocated to groups of up to 3 of the same sex but not randomised. MNT animals were randomised to groups of 6. Individual group weights were checked on first day of treatment prior to dosing to ensure they differed from the mean group weight by no more than 5%.
- Identification: Uniquely numbered ear-tags.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
1% Carboxymethylcellulose (CMC) in deionised water
Details on exposure:
Preparation:
Dosing preparations were freshly prepared prior to each dosing occasion. Formulations were protected from light and held at 15-25 °C and used withing 2 hours of preparation. Dose bottles were stirred continuosly immediately before and throughout dosing to ensure homogeneity.
Dose volume = 10 mL/kg
Frequency of treatment:
2 doses, approximately 24 hours apart
Post exposure period:
Bone marrow sampled 24 hours after final administration
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA) 20 mg/kg, was freshly repared in saline and administered only once via oral gavage 24 hours prior to necropsy.

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range finding experiment was performed on a group of three male and three female rats. The animals were treated with the test article at suitable doses until an estimate of the MTD was established. Animals were dosed once daily for two consecutive days with the test articles and observations made over a two day period following the final adminsitration. As no clinical signs of toxicity were observed at 2000 mg/kg bw/day, the main experiment was conducted using a max. dose of 2000 mg/kg bw/day.

TREATMENT AND SAMPLING TIMES:
The test article was given as two administrations, 24 hours apart and animals were sampled 24 hours after the final administration, thus enabling examination of cells exposed to the test article over a period of 24 to 48 hours prior to sampling.

DETAILS OF SLIDE PREPARATION:
Rats were killed by an overdose of sodium pentobarbitone i.p. and subsequent cervical dislocation. One femur from each animal was removed, cleaned and the ends removed from the shanks. Bone marrows were flushed from the marrow cavity with 2 mL foetal bovine serum into centrifuge tubes. 4 mL of serum were additionally added to each sample prior to adding to cellulose filtration columns (2 mL equal mix of type 50 and alpha cellulose). Once filtered, bone marrow cells were centrifuged and the pellets resuspended in serum. After a second centrifugation step, a drop of suspension was placed between two slides. A smear was made from the drop by drawing the end of a clean slide along the sample slide. After air-drying, slides were fixed for 10 min in absolute methanol and repeatedly rinsed in distilled water. Slides were allowed to dry and stained on the next day, after 10 min fixing in abs. methanol. After repeated rinsing, slides were stained for 5 min in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then allowed to dry and stored in the dark at room temperature prior to analysis.

METHOD OF ANALYSIS:
Scoring carried out using fluorescence microscopy. Relative proportions of PCE and normochromatic erythrocytes (NCE) were etermined until a total of at least 1000 cells (PCE plus NCE) had been analyzed. Then at least 2000 PCE per animal were examined for the presence of micronuclei.

Evaluation criteria:
For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data
3. The group mean MN PCE value at such a point exceeds the 95% calculated confidence interval for the mean historical vehicle control data
4. A dose-response trend in the proportion of MN PCE was observed (where more than two dose levels were analysed).

The assay was considered valid if all the following criteria were met:
1. The vehicle control MN PCE data were comparable with the laboratory’s historical vehicle control ranges
2. At least five animals out of each group were available for analysis, and
3. The positive control chemical (CPA) induced a statistically significant increase in the frequency of MN PCE.
Statistics:
After completion of microscopic analysis and decoding of the data the following were calculated:
1. % PCE for each animal and the mean for each group. The group mean % PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity
2. Frequency of MN PCE (i.e. MN per 2000 PCE) and % MN PCE for each animal and the group mean % MN PCE (± standard deviation).

The MN PCE data from the vehicle control group(s) were compared with the laboratory's historical vehicle control ranges (individual animal distribution data and calculated group mean 95% confidence interval) to determine whether the assay was acceptable. For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square test. The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square. Probability values of p ≤ 0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The assay data were considered valid.

No clinical signs of toxicity were observed in any animal following treatments with vehicle, Aluminium hydroxide (at 500, 1000 or 2000 mg/kg/day) or the positive control (CPA). No notable effect of treatment on bodyweights was observed.

Three individual %PCE values for the vehicle control dosed group were slightly higher than the historical mean %PCE values, but fell within the observed range for individual animals. Furthermore, the group mean % PCE value was within acceptable ranges and consistent with the historical vehicle control means. There was no evidence of any test article-induced toxicity to the bone marrow (as would usually be indicated by a notable decrease in %PCE values compared to the vehicle control group or dose dependant decrease).

Group mean frequencies of MN PCE were similar to and not statistically (chi-square) different from those seen in concurrent vehicle controls for all dose groups. Individual frequencies of MN PCE for all treated animals were consistent with historical vehicle control distribution data and similar to frequencies observed in recent historical controls.

Any other information on results incl. tables

Summary and Statistical Analysis of Micronucleus Data

Treatment (mg/kg/day) Cell Total % PCE Total MN PCE Mean MN PCE/2000 PCE % MN PCE SD Heterogeneity Contingency
X2 Significance X2C Significance
0 12000 49.35 16 24.504 0.13 0.10 46.204 NS    
500 12000 58.55 14 12.086 0.12 0.09 21.337 NS 0.03 NS
1000 12000 53.08 17 30.348 0.14 0.09 34.820 NS 0.00 NS
2000 12000 54.82 15 18.295 0.13 0.06 3.00 NS 0.00 NS
CPA, 20+ 12000 46.13 333 55.50 28.522 21.916     290.34 P£0.001

Linear trend: z = -0.043

NS Not significant

MN Micronucleated

+ Administered as a single dose

SD Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that Aluminium hydroxide did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to
2000 mg/kg/day (the maximum recommended dose for this study).
Executive summary:

Aluminium hydroxide was tested for its ability to induce micronuclei in the polychromatic erythrocytes (PCE) of the bone marrow of treated rats. In a range-finding study, no signs of toxicity were observed at 2000 mg/kg bw. Therefore, the animals were orally treated (gavage) with 500, 1000 and 2000 mg/kg bw of the test substance in the main study. Only male animals were treated in the main experiment as the range finder gave no indication of sex-specific differences regarding the effect of the test compound.

Rats treated with Aluminium hydroxide at all doses exhibited group mean %PCE that were similar to the vehicle control group and which were comparable with the laboratory’s observed historical control data, thus confirming there was no evidence of test article related bone marrow toxicity.

Rats treated with Aluminium hydroxide at all doses exhibited MN PCE frequencies that were similar to the vehicle control group and which were considered consistent with the laboratory's historical data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control. It is concluded that Aluminium hydroxide did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 2000 mg/kg/day (the maximum recommended dose for this study).