Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Jan 2007 - 17 Sep 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium dihydrogen triphosphate
EC Number:
237-714-9
EC Name:
Aluminium dihydrogen triphosphate
Cas Number:
13939-25-8
Molecular formula:
AlH2O10P3
IUPAC Name:
aluminium dihydrogen triphosphate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Triphosphoric acid aluminium salt
- Physical state: white powder
- Analytical purity: 90.8% (based on 60.8% P2O5 and 16.0% Al2O3)
- Impurities (identity and concentrations): based on XRD-Analysis, small amounts of Aluminium metaphosphate (Al(PO3)3) and Aluminium orthophosphate (AlPO4) are present (unknown concentration)
- Purity test date: July 2006 and re-analysis in April 2007
- Lot/batch No.: G460501F
- Stability under test conditions: After completion of the animal study, the test substance was analysed by the manufacturer and its stability was confirmed.
- Storage condition of test material: Cool dark place (measured value: 3-8°C), airtight

Test animals

Species:
mouse
Strain:
other: Crlj: CD1(ICR)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: ), Charles River Laboratories Japan, Atsugi Breeding Center
- Age at study initiation: 7 weeks on arrival, 8 weeks at dosing
- Weight at study initiation:
Preliminary study: 31.1-35.9 g (males), 23.5-28.3 g (females)
Main study: 32.6-36.9 g (males)
- Assigned to test groups randomly: no, under following basis: Animals were stratified using body weight on the day of group allocation (day of administration) and the mean body weight of each group was, as far as possible, equal. Individual allocation was carried out using a combination of the block assignment method and the random extraction method (the required groups were organised using the block assignment method and the study group and within group individual numbers are randomly allocated).
- Fasting period before study: no data
- Housing: no data
- Diet: CRF-1 (Oriental Yeast Co. Ltd.), ad libitum
- Water: Gotemba city tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 (preliminary study); 22-24 (main study)
- Humidity (%): 46-59 (preliminary study); 44-58 (main study)
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC-Na (carboxymethyl cellulose sodium) at 0.5% w/v in water for injection
- Justification for choice of solvent/vehicle: Selected in accordance with ‘Toxicity Study Guidelines’ because MMC is widely used in micronucleus tests and background data are available.
- Concentration of test material in vehicle:
Preliminary study: 25, 50, 100 and 200 mg/mL
Main study: 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): 4Y01
- Purity: in accordance with Japanese Pharmacopoeia
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in 0.5w/v% CMC-Na aqueous solution using a mortar to the prescribed concentration. Residual liquid in the test solution was absorbed onto a highly absorbable paper towel and incinerated.
The dose was 10 mL/kg body weight and mice were dosed using a flexible stomach sonde. Individual doses were calculated based on the body weight on the day of administration.
Duration of treatment / exposure:
Preliminary study: 24, 48 and 72 h
Main study: 24 h
Frequency of treatment:
Single administration
Post exposure period:
Not applicable
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250, 500, 1000 and 2000 mg/kg bw (preliminary study)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw (main study)
Basis:
actual ingested
No. of animals per sex per dose:
Preliminary study: 3 males and 3 females
Main study: 6 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Justification for choice of positive control(s): Selected in accordance with ‘Toxicity Study Guidelines’ because MMC is widely used in micronucleus tests and background data are available.
- Route of administration: intraperitoneal
- Doses / concentrations: The positive control was prepared as a 0.1 mg/mL solution in water for injection, and admnistered at 10 mL/kg bw to achieve a dose of 1 mg/kg bw

Examinations

Tissues and cell types examined:
Bome marrow cells from the femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The highest dose in the preliminary study was 2000mg/kg bw, which is the highest dose designated in the Toxicity Study Guidelines, this was divided by a common ratio of 2 and doses of 1000, 500 and 250mg/kg were established.
No mortality occurred and no signs of toxicity or a decrease in the number of immature erythrocytes were observed up to the highest dose.
Therefore, the dose levels in the main study were established at 500, 1000 and 2000 mg/kg bw.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): In the preliminary study, groups of 9 animals per sex and dose level were given the test material at 250, 500, 1000 and 2000 mg/kg bw by oral gavage. Groups of 3 animals per sex and dose level were sacrificed and bone marrow cells were collected at 24, 48 and 72 h post-administration, respectively.
In the main study, groups of 6 male animals per dose level were given the test material at 500, 1000 and 2000 mg/kg bw by oral gavage. Concurrent groups of 6 male animals each were given the vehicle (at 10 mL/kg bw by gavage) and the positive control (1 mg/kg bw, i.p. injection), respectively. All animals were sacreificed and bone marrow cells were collected at 24 h post-administration.

DETAILS OF SLIDE PREPARATION: Mice were euthanised at a prescribed time following administration by dislocation of the cervical vertebrae and bilateral femurs were then excised. Subsequently, bone marrow cells were flushed out into a centrifuge tube with about 0.1-0.2mL bovine foetal serum (GIBCO BRL, Lot No. 1299355 (preliminary study, main study) using a 1mL disposable syringe and a 23G needle. Bone marrow cells and foetal calf serum were then mixed using this syringe and syringe needle and cells were separated, centrifuged at 1000rpm for 5 minutes (Tomy Industries, benchtop centrifuge LC-220), the supernatant was discarded, the cell pellet was thoroughly mixed using a mixer and smeared on a slide glass (each smear sample was prepared from the left and right femurs of 1 animal). Smear samples were air dried, fixed in methanol (Wako Pure Chemical Industries, Lot No. EWQ0288 (preliminary study, main study)) for 3 minutes then air dried again. When samples were prepared, a comparison chart of the animal No. and sample No. was prepared, and a label showing the study No., stage, sample No./study type and preparation date was attached to the sample.

METHOD OF ANALYSIS: Observations were made blind based on the sample No. and 1 sample showing a favourable smear was selected. Acridine orange fluorescence staining and observations of bone marrow smear preparations were carried out. A bone marrow sample preparation was placed on a cover glass onto which a small amount of a 40µg/mL aqueous solution of acridine orange had been previously dropped. Observations were made at an excitation wavelength of around 490nm using a fluorescence microscope equipped with an observation filter transmitting at a wavelength of 515nm or more (System Biological Microscope, Olympus Optical, BX40 and a Universal epifluorescence microscope, Olympus, BX-FLA) at and total magnification of 600 times. Immature erythrocytes (PCE) and normochromatic erythrocytes were counted among a total of 200 erythrocytes per individual and the number of immature erythrocytes having micronuclei (MNPCE) among 2000 PCE were counted at the same time.
Evaluation criteria:
The test material was considered positive, if compared with the control values a statistically significant and dose-dependent increase in the frequency of micronucleated immature erythrocytes was observed.
Statistics:
The occurrence (%) and the number of micronucleated immature erythrocytes (MNPCE) compared to immature erythrocytes (PCE) per 2000 erythrocytes and the occurrence and number of PCE per 200 erythrocytes were obtained for 1 individual. The mean and SD were calculated for the number and occurrence (%) of MNPCE and the number and occurrence of PCE in each group and the maximum and minimum values for the occurrence were calculated.
In the main study, furthermore, after judgement of the significance of occurrence of micronuclei was confirmed to be within the mean ± 3 SD of the rate of occurrence of MNPCE in the negative control and positive control groups in the background data this laboratory, the negative group and test substance administration group were compared and the Kastenbaum & Bowman test (one-sided, p<0.05) and the Cochran Armitage test for trend (two-sided, p<0.05, 0.01) were carried out based on binomial distribution. For the occurrence of PCE, the F-test was used to investigate homoscedasticity (one-sided, p<0.05) between the negative control group and each test substance administration group, the Student t-test (two-sided, p<0.05, 0.01) was applied to the 1000mg/kg group as the distribution was equal and the Aspin t-test (two-sided, p<0.05, 0.01) was applied to the 500 and 2000mg/kg groups as distribution was unequal.
In the positive control group, the occurrence of MNPCE (%) was compared to the negative control group and the Kastenbaum & Bowman test (one-sided, p≤0.05) was carried out based on binomial distribution. Furthermore, for the occurrence of PCE too, homoscedasticity with the negative control group was investigated using the F-test (one-sided, p<0.05) and the Student t-test (two-sided, p<0.05, 0.01) was applied as the distribution was equal.

Results and discussion

Test resultsopen allclose all
Sex:
male/female
Genotoxicity:
negative
Remarks:
in the preliminary test
Toxicity:
no effects
Remarks:
in the preliminary test
Vehicle controls validity:
other: not examined in the preliminary test
Negative controls validity:
not examined
Positive controls validity:
other: not examined in the preliminary test
Sex:
male
Genotoxicity:
negative
Remarks:
in the main test
Toxicity:
no effects
Remarks:
in the main test
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000 and 2000 mg/kg bw
- Solubility: The test material was not soluble in the vehicle and was therefore applied as suspension.
- Clinical signs of toxicity in test animals: General conditions such as bodily appearance, nutritional status, behaviour and faeces were observed in both the preliminary and main studies in each group before administration, immediately after administration, about 2 hours after administration and once a day from the next day until the day of completion of bone marrow collection.
Body weights were measured on the day of administration and daily thereafter until the day of bone marrow collection.
No effects on general condition and body weight were observed.
- Evidence of cytotoxicity in tissue analyzed: No decrease in the number of immature erythrocytes was observed.
- Rationale for exposure: The route of administration was oral in accordance with the ‘Toxicity Study Guidelines’.
- Harvest times: 24, 48 and 72 h post-administration

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In the test substance administration groups, the occurrence of MNPCE was 0.09±0.09% in the 500mg/kg group, 0.10±0.03% in the 1000mg/kg group and 0.10±0.04% in the 2000mg/kg group. The results when these values were compared to 0.09±0.04% for the negative control group did not show a statistically significant increase (p≤0.05) in any administration group and a dose-dependent increase (p≤0.05, 0.01) was not seen.
The occurrence of PCE per 200 erythrocytes in each test substance administration group did not show a statistically significant decrease (p<0.05, 0.01) compared to the negative control group.
The occurrence of MNPCE in the positive control group was compared to the negative control group and showed a statistically significant increase. Furthermore, the occurrence of MNPCE in the negative control group and positive control group were within the mean ± SD of the background data in this laboratory (this institution).
- Ratio of PCE/NCE (for Micronucleus assay): The proportion (%) of PCE (including micronucleated PCE) was in the range of 52-64% in all test groups of the main study.
- Appropriateness of dose levels and route: The dose levels and route of administration were appropriately selected in accordance with the relevant Test Guideline.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, the test material did not induced a statistically significant and/or dose-dependent increase in the frequency of micronuclei in immature erythrocytes from bone marrow cells of Crlj: CD1(ICR) mice treated at up to 2000 mg/kg bw by the oral route. Therefore, the test material is considered to be not clastogenic in vivo.