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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following studies are provided for aluminium metaphosphate:

In vitro gene mutation in bacteria: negative with and without metabolic activation (OECD 471, GLP)

In vitro gene mutation in mammalian cells: negative with and without metabolic activation (OECD 476, GLP)

In vivo micronucleus study in cultured human peripheral lymphocytes: negative with and without metabolic activation (OECD 487, GLP)

Link to relevant study records

Referenceopen allclose all

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
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.
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
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
Target gene:
Thymidine kinase (Tk1)
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.
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
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

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.

Conclusions:
Aluminium metaphosphate did not cause genotoxic reactions in L5178Y mouse lymphoma cells with and without metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Oct 2014 - 17 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
313, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: distilled water was used as solvent/vehicle, since this medium is not suspected of chemical reaction with the test substance and is compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (10-50 µg/plate); benzo[a]pyrene (20 µg/plate)
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycin (6 µg/plate); sodium azide (1.5 µg/plate); ICR 191 acridine (1 µg/plate); 4-nitroquinoline-1-oxide (2 µg/plate)
Remarks:
without metabolic activation
Details on test system and experimental conditions:
INITIAL TEST
METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h
NUMBER OF REPLICATIONS: 3

CONFIRMATORY TEST
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48-72 h
NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies on the test plates compared to the solvent/vehicle control plates
Evaluation criteria:
In general, a 2 or 2.5-fold increase in the number of revertant colonies per plate over the background (spontaneous revertant frequency) is used as a criterion to distinguish active mutagens from non-mutagenic materials. The presence of dose response is a further criterion for mutagenic materials.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitation was seen at all test material concentrations, which did not influence the results of the assay

RANGE-FINDING/SCREENING STUDIES: A preliminary experiment was conducted to determine cytotoxicity and solubility of the test substance. Starting with the test material concentration of 5 mg/plate (recommended by OECD GL 471) and subsequent serial dilutions (ratio 1:2) following concentrations of test material were applied: 5, 2.5, 1.25, 0.625, 0.313 and 0.156 mg/plate on two bacteria strains (TA 98 and TA 1535) with different mutation type (frameshift and base-pair substitution) with and without metabolic activation.
The highest concentration of that solution was chosen for the main test that caused low cytotoxicity and/or where the precipitate did not interfere with the scoring according to OECD GL 471.

COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive control values were acceptable and compatible with the historical control values.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Test Material concentrations:

Concentration 1: 5 mg/plate

Concentration 2: 2.5 mg/plate

Concentration 3: 1.25 mg/plate

Concentration 4: 0.625 mg/plate

Concentration 5: 0.313 mg/plate

Table 2: Mean plate counts and standard deviations (SD); strain: TA98

 

Controls/ test substance conc.

Colony count

TA98

Main test:

Preincubation method

Confirmatory test:

Plate incorporation method

Mean

SD

Mean

SD

NK

18

3

11

4

NK+S9

22

4

18

3

PK

308

38

761

87

PK1+S9

866

44

2360

83

PK2+S9

167

42

198

58

Conc. 1

19

6

11

4

Conc. 1+S9

22

2

15

3

Conc. 2

19

4

10

2

Conc. 2+S9

21

5

21

10

Conc. 3

20

5

18

5

Conc. 3+S9

24

3

19

4

Conc. 4

20

1

13

5

Conc. 4+S9

24

9

18

7

Conc. 5

16

5

14

2

Conc. 5+S9

22

3

16

3

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (daunomycin)

PK1 - positive control 1 with S9 (2-aminoanthracene)

PK2 - positive control 2 with S9 (benzo(a)pyrene)

Conc. - concentration

 

Table 3: Mean plate counts and standard deviations (SD); strain: TA100

 

Controls/ test substance conc.

Colony count

TA100

Main test:

Preincubation method

Confirmatory test:

Plate incorporation method

Mean

SD

Mean

SD

NK

60

n. a.

60

6

NK+S9

81

9

69

15

PK

247

18

350

21

PK1+S9

944

61

1910

68

PK2+S9

507

40

806

44

Conc. 1

58

7

67

7

Conc. 1+S9

66

20

77

4

Conc. 2

53

13

71

9

Conc. 2+S9

72

11

83

12

Conc. 3

64

11

65

10

Conc. 3+S9

61

16

70

8

Conc. 4

54

6

64

8

Conc. 4+S9

73

6

71

9

Conc. 5

90

n. a.

73

1

Conc. 5+S9

91

13

69

8

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (sodium azide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

PK2 - positive control 2 with S9 (benzo(a)pyrene)

Conc. - concentration

n. a. - not applicable due to missing value for calculation

 

Table 4: Mean plate counts and standard deviations (SD); strain: TA1535

 

Controls/ test substance conc.

Colony count

TA1535

Main test:

Preincubation method

Confirmatory test:

Plate incorporation method

Mean

SD

Mean

SD

NK

6

3

5

n. a.

NK+S9

10

3

6

3

PK

349

18

371

19

PK1+S9

127

8

65

12

Conc. 1

8

2

11

2

Conc. 1+S9

9

n. a.

4

2

Conc. 2

5

2

6

4

Conc. 2+S9

10

n. a.

8

4

Conc. 3

8

2

6

4

Conc. 3+S9

5

n. a.

9

5

Conc. 4

5

1

3

2

Conc. 4+S9

5

2

7

4

Conc. 5

7

5

3

2

Conc. 5+S9

9

n. a.

9

n. a.

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (sodium azide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

n. a. - not applicable due to missing value for calculation

 

Table 5: Mean plate counts and standard deviations (SD); strain: TA1537

 

Controls/ test substance conc.

Colony count

TA1537

Main test:

Preincubation method

Confirmatory test:

Plate incorporation method

Mean

SD

Mean

SD

NK

4

1

3

1

NK+S9

6

1

3

3

PK

283

10

126

9

PK1+S9

210

32

107

13

Conc. 1

5

2

7

4

Conc. 1+S9

7

1

2

1

Conc. 2

8

3

5

1

Conc. 2+S9

5

1

4

1

Conc. 3

8

2

6

3

Conc. 3+S9

7

3

3

2

Conc. 4

6

2

5

1

Conc. 4+S9

5

2

4

3

Conc. 5

6

4

2

2

Conc. 5+S9

4

2

1

2

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (ICR 191 acridine)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

 

Table 6: Mean plate counts and standard deviations (SD); strain: WP2uvrA

 

Controls/ test substance conc.

Colony count

WP2uvrA

Main test:

Preincubation method

Confirmatory test:

Plate incorporation method

Mean

SD

Mean

SD

NK

17

3

16

3

NK+S9

18

3

20

6

PK

1419

129

1371

59

PK1+S9

309

26

407

55

Conc. 1

19

1

15

3

Conc. 1+S9

15

7

18

1

Conc. 2

19

5

20

5

Conc. 2+S9

16

1

23

4

Conc. 3

17

8

22

6

Conc. 3+S9

16

5

16

6

Conc. 4

18

4

17

5

Conc. 4+S9

10

2

16

4

Conc. 5

12

3

20

2

Conc. 5+S9

20

7

13

2

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (4-nitroquinoline-1-oxide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

Conclusions:
Under the conditions of this study, the test material did not induce reverse gene mutations in Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) or Escherichia coli (WP2 uvrA) tester strains. Therefore, the test material is considered to be not mutagenic in bacteria.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2015 - 04 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted September 26, 2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: C26967A
- Expiration date of the lot/batch: 27/06/2016
- Purity test date: 14/07/2015


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, in a tightly closed container and stored in a cool, dry and well-ventilated place.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test item was not soluble in any of the solvents recommended: aqueous media,
dimethylsulfoxide (DMSO), ethanol, acetone or 0.05 M H2SO4 solution). However, Aluminium metaphosphate was completely soluble as 3 μg per mL 0.05 M HCl solution by employing an ultrasonic bath (45 kHz) at 37°C for 10 minutes for preparing a solution. Hence, Aluminium metaphosphate was suspended in 0.05 M HCl solution for a concentration of 10 μg per mL medium. For concentrations lower than 10.0 μg/mL medium, the test item was completely dissolved in 0.05 M HCl.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Fresh preparations of the test item were prepared on the day of the experiment and used for the treatment in all experimental parts. No correction factor was used.
Species / strain / cell type:
lymphocytes: human peripheral lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human peripheral blood
- Suitability of cells: cells identified in OECD guideline.
- Sex, age and number of blood donors if applicable:Healthy non-smoking male of female individuals (18-35 years). No known recent exposures to genotoxic chemicals or radiation.
- Whether whole blood or separated lymphocytes were used if applicable:


Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.

Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
The concentration used for this assay was 5 μg/mL.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
In the preliminary experiment without and with metabolic activation concentrations of 0.01, 0.03162, 0.1, 0.3162, 1.0, 3.162 or 10.0 μg aluminium metaphosphate/mL medium were employed. Test item precipitation was noted at a concentration of 10.0 μg Aluminium metaphosphate/mL (24-hour or 4-hour exposure) in the experiment without and with metabolic activation.

Hence, the following concentrations were employed in the main study for the genotoxicity testswithout and with metabolic activation: 0.3162, 1.0, 3.162 or 10.0 μg Aluminium metaphosphate/mL medium.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: hydrochlorric acid (HCl)
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the solvents recommended: aqueous media, dimethylsulfoxide (DMSO), ethanol, acetone or 0.05 M H2SO4 solution). However, Aluminium metaphosphate was completely soluble as 3 μg per mL 0.05 M HCl solution by employing an ultrasonic bath (45 kHz) at 37°C for 10 minutes for preparing a solution.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Cholchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours and 24 hours at +37°C
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: 2 (main study), 1 (preliminary test)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm. The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide and left to air-dry.

NUMBER OF CELLS EVALUATED: The micronucleus frequencies were analysed in at least 2000 binucleate cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (relative increase in cell counts, RICC); cytotoxicity = 100% - RICC [%]

Evaluation criteria:
If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing micronuclei, it is classified as a positive result.
Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response.
Statistics:
The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971[1]) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity data).
Species / strain:
lymphocytes: human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- effects of pH / osmolality: No relevant changes in pH or osmolality of the test item formulations at
concentrations of 0.01 to 10.0 μg/mL medium were noted.
- Water solubility: substance was not soluble in water but was found to be soluble in HCl which was then used as the solvent in this test.
- Precipitation: In a preliminary study test item precipitation was noted at a concentration of 10 μg aluminium metaphosphate / ml (24 and 4 hour exposures). In the main study test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 10.0 μg test item/mL medium.


RANGE-FINDING/SCREENING STUDIES:In this preliminary experiment without and with metabolic activation concentrations of 0.01, 0.03162, 0.1, 0.3162, 1.0, 3.162 or 10.0 μg Aluminium metaphosphate/mL medium were employed. Test item precipitation was noted at a concentration of 10.0 μg Aluminium metaphosphate/mL (24-hour or 4-hour exposure) in the experiment without and with metabolic activation. Hence, 10.0 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (micronucleus frequencies per 1000 cells):
Mitomycin C:
Mean: 46.9
SD: 35
range 17-137
Colchicine:
Mean: 25.9
SD: 9.5
range 15-63
cyclophosphamide:
Mean: 44.0
SD: 37.7
range 14-158
- Negative (solvent/vehicle) historical control data:

Without metabolic activation
Untreated control
mean 6.6
SD 2.9
range 2.0 - 17
95% Confidence interval 5.9 - 7.3

Vehicle control
mean 6.3
SD 3.1
range 2.0 - 18
95% Confidence interval 5.7 - 6.8

With metabolic acitvation

Untreated control
mean 6.4
SD 2.6
range 3.0 - 13
95% Confidence interval 5.7 - 7.1

Vehicle control
mean 6.1
SD 4.2
range 1-22
95% Confidence interval 5.1 - 6.7

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI or RI in the case of the cytokinesis-block method

Table 1: Experiments without metabolic activation (S9 mix)

4 hr exposure

Concentration [µg/mL medium]

CBPI

RI [%]

Number of binucleate cells scored

Number of micronucleated cells per 1000 binucleate cells

0.05 M HCl

0

1.46

100

2000

5.0

Aluminium metaphosphate

0.3162

1.50

108

2000

5.0

1.0

1.50

108

2000

5.0

3.162

1.58

125

2000

8.0

10.0*

1.50

109

2000

4.5

Mitomycin C

0.2

1.40

87

2000

39.0 s.

 

24 hr exposure

Concentration [µg/mL medium]

CBPI

RI [%]

Number of binucleate cells scored

Number of micronucleated cells per 1000 binucleate cells

0.05 M HCl

0

1.43

100

2000

7.5

Aluminium metaphosphate

0.3162

1.35

82

2000

7.0

1.0

1.42

98

2000

7.5

3.162

1.36

85

2000

4.5

10.0*

1.43

102

1000

6.5

Colchicine

0.02

1.46

109

2000

28.0 s.

 

CBPI = Cytokinesis block proliferation index

RI = Replicative Index

s. = significantly different from negative control (p ≤ 0.05)

* = test item precipitation

Table 2: Experiment with metabolic activation (S9 mix)

4 hr exposure

Concentration [µg/mL medium]

CBPI

RI [%]

Number of binucleate cells scored

Number of micronucleated cells per 1000 binucleate cells

0.05 M HCl

0

1.47

100

2000

5.5

Aluminium metaphosphate

0.3162

1.49

106

2000

5.5

1.0

1.43

92

2000

4.5

3.162

1.40

86

2000

5.5

10.0*

1.51

109

2000

6.5

Cyclophosphamide

20

1.40

86

2000

22.5 s.

 

CBPI = Cytokinesis block proliferation index

RI = Replicative Index

s. = significantly different from negative control (p ≤ 0.05)

* = test item precipitation


 

Table 3: Experiment without metabolic activation (S9 mix) – 4 hr exposure

Culture number

Concentration [µg/mL medium]

Number of mononucleate cells per 500 cells scored

Number of binucleate cells per 500 cells scored

Number of multinucleate cells per 500 cells scored

CBPI

RI [%]

Number of binucleate cells scored

Number of micronucleated cells per 1000 binucleated cells

Significance chi2- test

0.05 M HCl

1

0

278

209

13

1.47

100

1000

6

-

11

0

290

196

14

1.45

100

1000

4

-

Aluminium metaphosphate

5

0.3162

282

199

19

1.47

100

1000

4

n.s.

15

0.3162

258

226

16

1.52

116

1000

6

n.s.

4

1.0

266

225

9

1.49

14

1000

5

n.s.

14

1.0

266

220

14

1.50

111

1000

5

n.s.

3

3.162

225

257

18

1.59

126

1000

7

n.s.

13

3.162

239

243

18

1.56

124

1000

9

n.s.

2

10.0*

274

219

7

1.47

100

1000

4

n.s.

12

10.0*

251

232

17

1.53

118

1000

5

n.s.

Mitomycin C

7

0.2

302

191

7

1.41

87

1000

36

s.

17

0.2

319

166

15

1.39

87

1000

42

s.

 

n.s. = not significantly different from negative control (p ≤ 0.05)

s. = significantly different from negative control (p ≤ 0.05)

CBPI = Cytokinesis block proliferation index

RI = Replicative test

*= test item precipitation

Table 4: Experiment without metabolic activation (S9 mix) – 24 hr exposure

Culture number

Concentration [µg/mL medium]

Number of mononucleate cells per 500 cells scored

Number of binucleate cells per 500 cells scored

Number of multinucleate cells per 500 cells scored

CBPI

RI [%]

Number of binucleate cells scored

Number of micronucleated cells per 1000 binucleated cells

Significance chi2- test

0.05 M HCl

1

0

312

186

2

1.38

100

1000

7

-

11

0

271

222

7

1.47

100

1000

8

-

Aluminium metaphosphate

15

0.3162

329

165

6

1.35

74

1000

6

n.s.

5

0.3162

335

160

5

1.34

89

1000

8

n.s.

4

1.0

307

187

6

1.40

105

1000

6

n.s.

14

1.0

290

205

5

1.43

91

1000

9

n.s.

3

3.162

346

148

6

1.32

84

1000

4

n.s.

13

3.162

306

189

5

1.40

85

1000

5

n.s

2

10.0*

279

217

4

1.45

118

1000

8

n.s.

12

10.0*

304

193

3

1.40

85

1000

5

n.s.

Colchicine

9

0.02

277

214

9

1.46

121

1000

26

s.

19

0.02

288

201

11

1.45

96

1000

30

s.

 

n.s = not significantly different from negative control (p ≤ 0.05)

s. = significantly different from negative control (p ≤ 0.05)

CBPI = Cytokinesis block proliferation index

RI = Replicative Index

*= test item precipitation

 

Table 5: Experiment with metabolic activation (S9 mix) – 4 hr exposure

Culture number

Concentration [µg/mL medium]

Number of mononucleate cells per 500 cells scored

Number of binucleate cells per 500 cells scored

Number of multinucleate cells per 500 cells scored

CBPI

RI [%]

Number of binucleate cells scored

Number of micronucleated cells per 1000 binucleated cells

Significance chi2- test

0.05M HCl

1

0

262

232

6

1.49

100

1000

6

-

9

0

286

209

5

1.44

100

1000

5

-

Aluminium metaphosphate

5

0.3162

259

228

13

1.51

104

1000

7

n.s.

13

0.3162

275

213

12

1.47

107

1000

4

n.s.

4

1.0

287

204

9

1.44

90

1000

3

n.s.

12

1.0

309

177

14

1.41

93

1000

6

n.s.

3

3.162

324

164

12

1.38

78

1000

4

n.s.

11

3.162

299

196

5

1.41

93

1000

7

n.s.

2

10.0*

257

231

12

1.51

104

1000

6

n.s.

10

1.0*

267

214

19

1.50

114

1000

7

n.s.

Cyclophosphamide

7

20

333

160

7

1.35

71

1000

24

s.

15

20

295

191

14

1.44

100

1000

21

s.

 

n.s. = not significantly different from negative control (p ≤ 0.05)

s. = significantly different from negative control (p ≤ 0.05)

CBPI = Cytokinesis block proliferation index

RI replicative index

*= test item precipitation

Conclusions:
Under the present test conditions, Aluminium metaphosphate tested up to a concentration of 10.0 μg/ml medium that led to test item precipitation in the absence
and in the presence of metabolic activation employing two exposure times (4 and 24 h, without S9) and one exposure time (4 h, with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Aluminium metaphosphate is not genotoxic and therefore no mode of action is identified.

Additional information

Justification for classification or non-classification

No classification for in vitro genetic toxicity is proposed for aluminium metaphosphate. The data has been generated according to the recommended guidelines and under the conditions of GLP, this data is considered to be adequate for the purposes of classification and labelling in accordance with Regulation (EC) No.1272/2008 (EU CLP) and GHS.