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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 07 January 2010 and 05 March 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no deviations from standard test guidelines and no methodological deficiencies. This study is conducted according to an appropriate guideline and under the conditions of GLP, the study is therefore considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for Regulation (EC) No. 1907/2006 (REACH). The reliability has been amended in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. The study is considered to be adequate and reliable for the purpose of registration under REACH (Regulation (EC) No. 1907/2006) and for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). Read-across is justified on the basis that the genotoxic potential of aluminium dihydrogen triphosphate will be determined by the Al cation. Triphosphate itself is not considered to be a genotoxic as the ultimate breakdown product of triphosphate (orthophosphate) is routinely used in the nutrient broths (usually as sodium or potassium orthophosphate) that cell colonies as well as in S9-mix in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. As aluminium dihydrogen triphosphate has a lower water solubility than aluminium orthophosphate it is considered to be less bioavailable and therefore aluminium orthophosphate is considered to be a worst case for genotoxic potential of the Al cation. The study reports that aluminium orthophosphate is a not genotoxic under the conditions of the study.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 15th September 2009, Date of signature: 26th November 2009
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Aluminium orthophosphate
EC Number:
232-056-9
EC Name:
Aluminium orthophosphate
Cas Number:
7784-30-7
IUPAC Name:
[phosphato(3-)-kappa~3~O,O',O'']aluminum
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Sponsor's identification: Aluminium orthophosphate
Description : White powder
Chemical name: Tribasic aluminium phosphate (M13-03)
Purity : 99.7%
Batch number : A88770A
Date received : 28 August 2009
Expiry date : 05 June 2010
Storage conditions: Room temperature in the dark

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Experiment 1:
Group Final concentration of Aluminium orthophosphate (µg/ml)
4(20)-hour without S9 0*, 20.0, 40.0, 80.0*, 160.0*, 320.0*, 480.0, MMC 0.4*
4(20)-hour with S9 0*, 20.0, 40.0, 80.0*, 160.0*, 320.0*, 480.0, CP 5*
24-hour without S9 0*, 20.0, 40.0, 80.0*, 160.0*, 320.0*, 480.0, MMC 0.2*
* Dose levels selected for metaphase analysis

MMC = Mitomycin C
CP = Cyclophosphamide
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: MEM
- Justification for choice of solvent/vehicle: MEM was selected as the solvent because the test material was readily soluble in it at the required concentrations.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9

Migrated to IUCLID6: (CP)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9

Migrated to IUCLID6: (MMC)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium):
20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hrs.

SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS:
Duplicate cultures

NUMBER OF CELLS EVALUATED:
100/culture

DETERMINATION OF CYTOTOXICITY
- Method:
mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells

OTHER:
None.

Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Refer to information on results and attached tables.
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: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material suspended in MEM
- Precipitation: SEE PRELIMINARY TOXICITY TEST

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 4.9 to 1250 µg/ml. The molecular weight of the test material was supplied as 125 and therefore the maximum recommended dose level was 1250 µg/ml, which was equivalent to 10 mM. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure at and above 156.3 µg/ml in the 4(20)-hour and 24-hour exposure groups in the absence of metabolic activation (S9), and at and above 39.1 µg/ml in the 4(20)-hour exposure with S9. However, it should be noted that precipitate was observed in the blood pellet in the 24-hour exposure group at and above 19.5 µg/ml. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1250 µg/ml in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 1250 µg/ml. It should be noted that precipitate persisted onto the slides at and above 312.5 µg/ml in the 4(20)-hour exposure groups, and at and above 156.3 µg/ml in the 24-hour exposure group. The mitotic index data are presented in Table 1. The test material induced some evidence of toxicity in the 4(20) hour exposure in the presence of metabolic activation (S9) only. The selection of the maximum dose level was based on precipitate observations (either cloudy or more pronounced form) and was 480.0 µg/ml for both the pulse and continuous exposure groups.


COMPARISON WITH HISTORICAL CONTROL DATA: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
EXPERIMENT 1:
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Aluminium orthophosphate (µg/ml)
4(20)-hour without S9 0*, 20.0, 40.0, 80.0*, 160.0*, 320.0*, 480.0, MMC 0.4*
4(20)-hour with S9 0*, 20.0, 40.0, 80.0*, 160.0*, 320.0*, 480.0, CP 5*
24-hour without S9 0*, 20.0, 40.0, 80.0*, 160.0*, 320.0*, 480.0, MMC 0.2*
* Dose levels selected for metaphase analysis

MMC = Mitomycin C
CP = Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum dose level of test material (480.0 µg/ml) in all three exposure groups.
Precipitate observations were taken and a precipitate was noted in the 4(20)-hour exposure groups at the end of exposure at and above 20.0 µg/ml, and at and above 320 µg/ml at harvest. In the 24-hour exposure group precipitate was observed at and above 40 µg/ml at the end of exposure.

The mitotic index data are given in Table 2 and Table 3. They confirm the qualitative observations in that no inhibition of mitotic index was observed in any exposure group.
The maximum dose level selected for metaphase analysis was 320.0 µg/ml), which was limited by precipitate.
The chromosome aberration data are given in Table 4, Table 5 and Table 6. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the sensitivity of the assay and the efficacy of the metabolising system.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The polyploid cell frequency data are given in Table 7. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.
There was no evidence of a response in the presence of metabolic activation in this study or in the Mouse Lymphoma Assay (MLA) using L5178Y cells performed on the test material (Harlan Laboratories Ltd Project Number 2920/0085). This was taken as evidence to confirm scientific justification that the repeat of the exposure group with metabolic action was not required.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Historical Aberration Ranges for Vehicle Control Cultures

Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures. The current in-house historical aberration ranges are presented below:


 

 

Pulse exposure –S9

Pulse exposure +S9

(1% S9)

24-Hour –S9

Pulse exposure +S9

(2% S9)

 

% cells with aberrations (-gaps)

% cells with polyploids

% cells with aberrations (-gaps)

% cells with polyploids

% cells with aberrations (-gaps)

% cells with polyploids

% cells with aberrations (-gaps)

% cells with polyploids

Minimum

0

0

0

0

0

0

0

0

Maximum

2.5

1

2

0.5

2.5

0.5

2.0

0.5

Mean

0.73

0.06

0.53

0.02

0.53

0.03

0.52

0.05

Standard Deviation

0.71

0.21

0.49

0.09

0.68

0.12

0.57

0.16

Number

32

32

32

32

32

32

28

28

2.2      Historical Aberration Range for Positive Control Cultures

 

Pulse exposure –S9

MMC 0.4 µg/ml

Pulse exposure +S9

(1% S9)

CP 10 - 12.5 µg/ml

24-Hour –S9

MMC 0.2 - 0.25 µg/ml

Pulse exposure +S9

(2% S9)

CP 10 - 12.5 µg/ml

 

% cells with aberrations (-gaps)

% cells with polyploids

% cells with aberrations (-gaps)

% cells with polyploids

% cells with aberrations (-gaps)

% cells with polyploids

% cells with aberrations (-gaps)

% cells with polyploids

Minimum

7.5

0

10.0

0

6.5

0

8.0

0

Maximum

62.0

0.7

49.0

1

67.0

1.0

38.0

1.0

Mean

34.79

0.02

25.80

0.12

37.79

0.05

22.38

0.04

Standard Deviation

15.79

0.12

8.56

0.29

16.62

0.20

7.62

0.19

Number

32

32

32

32

32

32

27

27

For the tables and figures of resluts mentioned above, please refer to the attached background material section for the following tables:

Table 1: Mitotic Index - Preliminary Toxicity Test

Table 2: Mitotic Index - Experiment 1

Table 4: Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9)

Table 5: Results of Chromosome Aberration Test - Experiment 1 With Metabolic Activation (S9)

Table 6: Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9)

Table 7:  Mean Frequency of Polyploid Cells (%)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

The study is considered to be of sufficient adequacy, reliability and relevance to be used as a key study for this endpoint.
Read-across is justified on the basis that the genotoxic potential of aluminium dihydrogen triphosphate will be determined by the Al cation. Triphosphate itself is not considered to be a genotoxic as the ultimate breakdown product of triphosphate (orthophosphate) is routinely used in the nutrient broths (usually as sodium or potassium orthophosphate) that cell colonies as well as in S9-mix in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates.

As aluminium dihydrogen triphosphate has a lower water solubility than aluminium orthophosphate it is considered to be less bioavailable and therefore aluminium orthophosphate is considered to be a worst case for genotoxic potential of the Al cation. The study reports that aluminium orthophosphate is a not genotoxic under the conditions of the study.