Aluminium metaphosphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26/08/2014 - 04/12/2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Performed in 2014. This study is now covered by the OECD 490 guideline.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Guideline was not available in 2014 when the study was performed

GLP compliance:

yes

Type of assay:

other: gene mutation in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium metaphosphate

EC Number:

237-415-3

EC Name:

Aluminium metaphosphate

Cas Number:

13776-88-0

Molecular formula:

Al.3HO3P

IUPAC Name:

aluminium metaphosphate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: C08567A
- Expiration date of the lot/batch: 23/01/2016
- Analytical purity: >95%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature 15-25°C
- Stability under test conditions: stable


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: sterilised via gamma irradiation

FORM AS APPLIED IN THE TEST (if different from that of starting material)
In suspension

Method

Target gene:

Thymidine kinase (Tk1)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Bayer Schering Pharma AG cell line L5178Y TK+/- 3.7.2C
- Suitability of cells: Appropriate cells used in accordance with the guideline

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cell culture medium (RPMI 1640) containing 10% v/v horse serum. 5% CO2.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

5 mg/l
1 mg/ml
0.2 mg/ml
0.04 mg/ml
0.008 mg/ml
0.0016 mg/ml
0.00032 mg/ml
Top dose is recommended by OECD GL 476.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium (RPMI 1640) containing 10% v/v horse serum
- Justification for choice of solvent/vehicle: medium is not suspected of chemical reaction with the test substance and is compatible with the survivial of the cells and the S9 activity.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 0.5 x 1E6/ml

DURATION
- Exposure duration: 3 hours (first test) 4 hours (confirmatory test)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent):11-14 days (with TFT)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: cloning efficiency
- Any supplementary information relevant to cytotoxicity: cloning for cloning effeciency was performed after the exposure time (CE1) and after the expression period (CE2).
CE1: each treated cell culture was diluted to 8 cells/ml = 1.6 cells per well with RPMI 1640 containing 20% v/v HS and cultivated in a humidified incubator with 5% CO2 in air at 37±1°C for 7 days.
CE2: An aliquot of each treated cell culture was diluted to 8 cells/ml - 1.6 cells per well with RPMI 1640 + 20% v/v HS and cultivated in a humidifier incubator with 5% CO2 in air at 37±1°C for 7 days.

Rationale for test conditions:

Testing was performed in accordance with the guideline.

Evaluation criteria:

The cloning efficiency (CE) of cells and the spontaneous mutation frequency (MF) of negative controls should be within their limit valuesL CE ≥ 50%; MF (negative control) 50-300 x 10E-6.
The MF of positive controls should be increased 2x or more than the MF in corresponding negative controls in each experiment.

Statistics:

Not specified

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight precipitations were seen in all test material concentrations ehich did not influence the results of the assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH did not change during the study

Mutation frequencies were determined from the number of wells without colonies, without big colonies(≥1/4 of well diameter, large and diffuse morphology), without small colonies (≤1/4 of the well diameter, compact morphology). A significant increase in mutation frequencies (2 x higher than the corresponding negative controls) was only found in positive controls.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
CE1:
Negative control (-S9): Min 0.88 - Max 1.25 (mean 0.84). SD: 0.15 Number of studies = 5
Negative control (+S9): Min 0.87 - Max 1.05 (mean 0.87). SD: 0.09 Number of studies = 5
Positive control (-S9): Min 0.63 - Max 1.01 (mean 1.04). SD: 0.14 Number of studies = 5
Positive control (+S9): Min 0.80 - Max 1.00 (mean 0.99). SD: 0.13 Number of studies = 5

CE2:
Negative control (-S9): Min 0.90 - Max 1.22 (mean 0.86). SD: 0.17 Number of studies = 5
Negative control (+S9): Min 0.93 - Max 1.24 (mean 0.85). SD: 0.16 Number of studies = 5
Positive control (-S9): Min 0.61 - Max 1.08 (mean 1.16). SD: 0.27 Number of studies = 5
Positive control (+S9): Min 0.73 - Max 1.12 (mean 1.06). SD: 0.11 Number of studies = 5

MF (big and small colonies):
Negative control (-S9): Min 61.13 - Max 115.14 (mean 676.57). SD: 168.46 Number of studies = 5
Negative control (+S9): Min 99.47 - Max 116.59 (mean 685.85). SD: 133.27 Number of studies = 5
Positive control (-S9): Min 496.35 - Max 900.51 (mean 94.05). SD: 19.75 Number of studies = 5
Positive control (+S9): Min 547.86 - Max 866.44 (mean 107.86). SD: 7.52 Number of studies = 5

Any other information on results incl. tables

MF (main test):

	MF
	Big colonies	Small colonies
Negative control (-S9)	109.93	64.38
Negative control (+S9)	124.78	68.03
Positive control (-S9)	329.11	1854.44
Positive control (+S9)	313.13	501.96
Concentration 1 (-S9)	106.04	87.39
Concentration 1 (+S9)	113.14	100.13
Concentration 2 (-S9)	115.50	88.29
Concentration 2 (+S9)	124.31	65.67
	MF
	Big colonies	Small colonies
Negative control (-S9)	96.68	51.93
Negative control (+S9)	76.79	39.62
Positive control (-S9)	358.67	719.25
Positive control (+S9)	205.27	419.71
Concentration 3 (-S9)	91.98	48.45
Concentration 3 (+S9)	93.99	70.05
Concentration 4 (-S9)	93.11	44.58
Concentration 4 (+S9)	102.78	46.75

 

MF confirmatory test

	MF
	Big colonies	Small colonies
Negative control (-S9)	94.33	72.65
Negative control (+S9)	94.09	81.38
Positive control (-S9)	443.45	1571.21
Positive control (+S9)	195.30	227.77
Concentration 1 (-S9)	79.73	58.03
Concentration 1 (+S9)	99.79	57.48
Concentration 2 (-S9)	92.75	70.09
Concentration 2 (+S9)	118.78	52.94
Concentration 3 (-S9)	88.45	58.94
Concentration 3 (+S9)	102.01	59.38
Concentration 4 (-S9)	104.23	58.96
Concentration 4 (+S9)	111.06	62.99

The variability of the MF of all tested concentrations are as a result of biological variance of the test system and displayed not significant increase.

The presence of S9 had no effect on the results.

The distribution of smal and big colonies showed neither chromosome nor gene mutations.

Applicant's summary and conclusion

Conclusions:

Aluminium metaphosphate did not cause genotoxic reactions in L5178Y mouse lymphoma cells with and without metabolic activation.