Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data is available for magnesium hydrogenorthophosphate. Read across data with trimagensium bis(orthophosphate) tetrahydrate (CAS 13465 -22 -0) is used and considered reliable.

In vitro:

Gene mutation (Bacterial reverse mutation assay / Ames test; RA-A 13465 -22 -0): S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. Coli WP2 uvrA were all negative with and without metabolic activation (OECD 471)

Cytogenicity (chromosome aberration test, RA-A 13465 -22 -0): negative (OECD 473)

Gene mutation (mammalian cells / mouse lymphoma assay, RA-A 13465 -22 -0): negative (OECD 476)

In vivo:

no data available and no further data needed

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to read across justification in IUCLID chapter 13
Reason / purpose:
read-across source
Key result
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:
valid
Positive controls validity:
valid
Key result
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
-Dimethyl suphoxide solubility: The test material was fully soluble in sdimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Preliminary Toxicity Test

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without

(-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

122

115

116

128

115

106

119

104

107

100

110

+

TA100

94

91

99

104

71

90

97

91

98

88

84

-

WP2uvrA-

17

32

25

16

28

32

25

18

19

27

29

+

WP2uvrA-

30

33

24

28

31

27

22

32

29

24

32

In the range-finding test (Experiment 1 – plate incorporation method) the test material caused no visible reduction in the growth of the bacterial background lawn at any dose level in either the absence or presence of S9-mix. However, in the second experiment (pre-incubation methodology) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester stains dosed in the absence of S9-mix at 5000 µg/plate. No toxicity was noted to any of the bacterial strains dosed in the presence of S9-mix. The toxicity observed was of insufficient severity to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation for the Main test are presented in the tables below:

Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

104

 

21

 

19

 

19

 

9

 

104

(101)

20

(24)

21

(18)

9

(16)

13

(13)

95

 

32

 

13

 

21

 

16

 

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

104

 

20

 

19

 

22

 

12

 

102

(101)

28

(22)

25

(25)

16

(19)

11

(11)

98

 

19

 

30

 

19

 

10

 

Table 2 Test Results: Range-Finding Test– Without Metabolic Activation

Test period

From: 30 January 2010

To: 02 February 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

88

92

109

(96)

11.2#

20

23

23

(22)

1.7

16

16

22

(18)

3.5

21

18

18

(19)

1.7

11

14

11

(12)

1.7

-

50

82

70

85

(79)

7.9

20

16

19

(18)

2.1

13

24

19

(19)

5.5

13

12

14

(13)

1.0

13

9

12

(11)

2.1

-

150

85

84

89

(86)

2.6

16

21

15

(17)

3.2

16

22

14

(17)

4.2

14

14

19

(16)

2.9

13

8

13

(11)

2.9

-

500

85

77

95

(86)

9.0

19

16

16

(17)

1.7

11

22

19

(17)

5.7

12

16

14

(14)

2.0

11

13

12

(12)

1.0

-

1500

88

90

86

(88)

2.0

14

24

24

(21)

5.8

12

12

20

(15)

4.6

12

12

12

(12)

0.0

13

11

15

(13)

2.0

-

5000

87

93

104

(95)

8.6

20

15

16

(17)

2.6

16

16

23

(18)

4.0

15

20

15

(17)

2.9

8

9

12

(10)

2.1

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

607

517

531

(552)

48.4

1683

1649

1740

(1691)

46.0

397

428

428

(418)

17.9

113

118

121

(117)

4.0

544

534

551

(543)

8.5

ENNG4NQO9AA#

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Table 3 Test Results: Range-Finding Test– With Metabolic Activation

Test period

From: 30 January 2010

To: 02 February 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

99

102

97

(99)

2.5#

12

12

10

(11)

1.2

20

19

18

(19)

1.0

16

24

23

(21)

4.4

11

15

13

(13)

2.0

+

50

88

91

109

(96)

11.4

9

9

11

(10)

1.2

13

18

23

(18)

5.0

17

24

15

(19)

4.7

8

15

14

(12)

3.8

+

150

84

74

81

(80)

5.1

12

12

11

(12)

0.6

19

15

18

(17)

2.1

17

15

19

(17)

2.0

13

15

14

(14)

1.0

+

500

75

76

81

(77)

3.2

9

9

7

(8)

1.2

24

16

16

(19)

4.6

20

19

18

(19)

1.0

13

13

12

(13)

0.6

+

1500

75

85

71

(77)

7.2

9

9

12

(10)

1.7

21

16

16

(18)

2.9

18

22

15

(18)

3.5

15

16

12

(14)

2.1

+

5000

80

79

95

(85)

9.0

8

14

13

(12)

3.2

11

25

24

(20)

7.8

16

18

17

(17)

1.0

11

14

16

(14)

2.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1052

1258

1057

(1122)

117.5

242

213

217

(224)

15.7

166

186

223

(192)

28.9

244

260

289

(264)

22.8

143

231

96

(157)

68.5


BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

#       Standard deviation

Table 4 Test Results: Main Test– Without Metabolic Activation

Test Period

From: 04 February 2010

To: 07 February 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

109

113

80

(101)

18.0#

17

19

19

(18)

1.2

28

17

17

(21)

6.4

22

24

21

(22)

1.5

8

13

13

(11)

2.9

-

50

92

87

97

(92)

5.0

22

21

21

(21)

0.6

21

21

21

(21)

0.0

22

22

20

(21)

1.2

14

12

12

(13)

1.2

-

150

100

105

81

(95)

12.7

17

20

18

(18)

1.5

16

16

21

(18)

2.9

19

19

15

(18)

2.3

11

12

12

(12)

0.6

-

500

111

90

94

(98)

11.2

16

21

21

(19)

2.9

21

20

20

(20)

0.6

21

25

19

(22)

3.1

14

11

11

(12)

1.7

-

1500

110

96

113

(106)

9.1

16

21

17

(18)

2.6

24

15

24

(21)

5.2

18

17

17

(17)

0.6

13

13

12

(13)

0.6

-

5000

104 *

109 *

104 *

(106)

2.9

21 *

21 *

17 *

(20)

2.3

22 *

22 *

18 *

(21)

2.3

20 *

17 *

18 *

(18)

1.5

13 *

12 *

13 *

(13)

0.6

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

515

613

520

(549)

55.2

359

359

365

(361)

3.5

671

690

686

(682)

10.0

153

116

146

(138)

19.7

1012

888

1019

(973)

73.7

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

*         Partial absence of bacterial background lawn

#        Standard deviation

Conclusions:
Interpretation of results:
negative

Magnesium hydrogenorthophosphate was considered to be non-mutagenic under the conditions of this test.
Executive summary:

No genotoxic potential was seen from the gene mutation test in bacteria of the source substance trimagnesium bis(orthophosphate) tetrahydrate. As explained in the justification for type of information, the differences in molecular structure between magnesium hydrogenorthophosphate and trimagnesium bis(orthophosphate) tetrahydrate are unlikely to lead to differences in the Ames test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to read across justification in IUCLID chapter 13
Reason / purpose:
read-across source
Key result
Species / strain:
lymphocytes: Human
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: 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:
Premlinary toxictiy test: The dose range for the Preliminary Toxicity Test was 13.0 to 3340 μg/ml. The molecular weight of the test material was supplied as 334 and therefore the maximum recommended dose level was 3340 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 52.2 μg/ml, in the 4(20)-hour pulse exposure groups and at and above 208.8 μg/ml in t e 24-hour continuous exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 3340 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 3340 μg/ml. The mitotic index data are presented in Table 1. The test material induced no evidence of toxicity in any of the exposure groups. With no clear toxicity the selection of the maximum dose level for the main experiment was based on the lowest precipitating dose level and was 1670 μg/ml for both the 4(20)-hour pulse exposure groups and 24-hour continuous exposure group.


RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 19.53 to 5000 µg/ml. The maximum dose was the maximum recommended dose level. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure period at all dose levels of the test material in both the 4(20)-hour exposure groups. In the 24-hour continuous exposure group precipitate was observed at and above 39.06 µg/ml in the parallel blood-free cultures although it was seen at all dose levels in the blood cultures at the end of exposure. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 µg/ml in all three exposure groups although there was toxicity noted in the 24-hour continuous exposure group with reduced numbers of metaphases observed at the maximum dose. Precipitate was present on the slides at all dose levels of the test material, although it did not interfere with the assessment of the metaphases it may account for some variability in the mitotic index data as its presence makes scoring more difficult. The mitotic index data are presented in Table 1 (see attached background material). The test material induced evidence of toxicity in the 24-hour continuous exposure group but only showed moderate toxicity in the 4(20)-hour exposure groups.
The selection of the maximum dose level was based on the maximum recommended dose level and the maximum dose level was 5000 µg/ml for the 4(20)-hour exposure groups. For the 24-hour continuous exposure group used in Experiment 2 the maximum dose was limited by toxicity, and was 1250 µg/ml.


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.

MAIN TEST:
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 1670 μg/ml in all three exposure groups. Precipitate observations were taken and were similar to those taken in the Preliminary Toxicity Test, and a precipitate of the test material was noted at and above 208.8 μg/ml. 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 1670 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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. 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 8. 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 MLA test performed in conjunction on the test material (Harlan Project No. 2920/0082) which can also detect clastogenic activity. In the OECD 473 Guideline, it is recommended that a repeat of the 4(20)-hour exposure with metabolic activation is performed if a negative response is seen in the first experiment, unless there is scientific justification for its omission. The quoted study was performed to meet the requirements of the OECD 476 Guideline and was negative. It was, therefore, considered that referencing this study gave adequate scientific justification for the omission of the repeat of the with metabolic activation exposure group.

Deviation from the test guideline:

In the OECD 473 Guideline, it is recommended that a repeat of the 4(20)-hour exposure with metabolic activation is performed if a negative response is seen in the first experiment, unless there is scientific justification for its omission. This study was run in conjunction with a which has the capability of detecting clastogenic activity. The quoted study was performed to meet the requirements of the OECD 476 Guideline. It was, therefore, considered that referencing a study, such as the Mouse Lymphoma Assay (MLA) using L5178Y cells, gave adequate scientific justification for the omission of the repeat of the with metabolic activation exposure group.

See attached document for tables

Conclusions:
Interpretation of results:
negative with and without metabolic activation


Magnesium hydrogenorthophosphate is assumed to 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. Magnesium hydrogenorthophosphate was therefore considered to be non-clastogenic to human lymphocytes in vitro.

This study has been selected as the key study because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation EC (No.) 1272/2008 (EU CLP).
Executive summary:

No genotoxic potential was seen from the chromosome aberration assay of the source substance trimagnesium bis(orthophosphate) tetrahydrate. As explained in the justification for type of information, the differences in molecular structure between magnesium hydrogenorthophosphate and trimagnesium bis(orthophosphate) tetrahydrate are unlikely to lead to differences in the chromosome aberration assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to read across justification in IUCLID chapter 13
Reason / purpose:
read-across source
Statistics:
n.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS

Preliminary Toxicity Test

The dose range of the test material used in the preliminary toxicity test was 16.38 to 4192 µg/ml

In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of a modest reduction in the relative suspension growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of metabolic activation there was evidence of a marked reduction in %RSGvalues of cells treated with test material. A precipitate of the test material was observed at and above 32.75 µg/ml in the 4-hour exposure groups, and at and above 262 µg/ml in the 24-hour exposure group. In the subsequent mutagenicity test, the maximum dose level for all three of the exposure groups was 4192 µg/ml.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

4-Hour Exposure With and Without Metabolic Activation

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

As was seen in the preliminary toxicity test, there was evidence of modest dose related toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the % RSG and RTG values (Tables 3 and 6). There was no evidence of any reductions in viability (%V), therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). A precipitate of test material was observed at and above 262 µg/mL.

The numbers of small and large colonies and their analysis are presented in attached Tables 4 and 7.

24-Hour Exposure Without Metabolic Activation

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 10.

As was seen in the preliminary toxicity test, once again there was evidence of marked dose related toxicity following exposure to the test material, as indicated by the %RSGand RTG values (Table 9). There was no evidence of any reductions in viability (%V), therefore indicating that no residual toxicity had occurred. Near optimum levels of toxicity were achieved. The positive control induced acceptable levels of toxicity (Table 9).
The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test material.
The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10-6viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily (Table 9).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell (Table 9). A precipitate of test material was observed at and above 262 µg/mL.

The numbers of small and large colonies and their analysis are presented in attached Table 10.

Please see Attached "Tables 1 to 10"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:
Interpretation of results:
negative, non-mutagenic

Magnesium hydrogenorthophosphate is assumed to not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test. This study has been selected as the key study because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).
Executive summary:

No genotoxic potential was seen from the mouse lymphoma assay of the source substance trimagnesium bis(orthophosphate) tetrahydrate. As explained in the justification for type of information, the differences in molecular structure between magnesium hydrogenorthophosphate and trimagnesium bis(orthophosphate) tetrahydrate are unlikely to lead to differences in the mouse lymphoma assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Since all in vitro genetic toxicity test were negative no further in vivo testing is required according to Annex VIII (8.4) of the REACh regulation.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No studies are available with magensium hydrogenorthophosphate (CAS 7757 -86 -0). Reliable data is available for trimagensium bis(orthophosphate) tetrahydrate (CAS 13465 -22 -0).

Magnesium hydrogenorthophosphate and trimagensium bis(orthophosphate) tetrahydrate are strucutrally similar ionic compounds with similar low systemic toxicity suggesting that they are metabolised via similar metabolic pathways and to similar breakdown products. Therefore, a similar gentoxic potential is assumed.

Genetic toxicity of trimagensium bis(orthophosphate)-4 hydrate was evaluated in three different in vitro tests.

Genetic toxicity (mutagenicity) in bacteria in vitro:

A GLP guidline according to OECD 471 is available with trimagnesium bis(orthophosphate) 4 -hydrate (Harlan, 2010).

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The concentrations used were 50 to 5000 µg/plate.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the range-finding test (Experiment 1 – plate incorporation method) the test material caused no visible reduction in the growth of the bacterial background lawn at any dose level in either the absence or presence of S9-mix. However, in the second experiment (pre-incubation methodology) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester stains dosed in the absence of S9-mix at 5000 µg/plate. No toxicity was noted to any of the bacterial strains dosed in the presence of S9-mix. The toxicity observed was of insufficient severity to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. In conclusion, trimagnesium bis(orthophosphate) 4 -hydrate and thus magensium hydrogenorthophosphate are considered to be non-mutagenic under the conditions of this test.

Genetic toxicity (cytogenicity) in mammalian cells in vitro:

A GLP guidline according to OECD 473 is available with trimagnesium bis(orthophosphate) 4 -hydrate (Harlan, 2010).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to three dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, i.e. 4 hours= exposure in the absence of metabolic activation (S9) with a 20-hour expression period, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24 hours continuous exposure in the absence of metabolic activation.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All 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 was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that was limited by precipitate.

In conclusion, trimagnesium bis(orthophosphate) tetrahydrate and thus magensium hydrogenorthophosphate are considered to be non-clastogenic to human lymphocytes in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro:

A GLP guidline according to OECD 476 is available with trimagnesium bis(orthophosphate) 4 -hydrate (Harlan, 2010)

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (R0 medium) and positive controls. The exposure groups used were as follows: 4‑hour exposures both with and without metabolic activation, and 24 hours without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 262 to 4192 µg/mL for all three of the exposure groups. 

The maximum dose level used was equivalent to approximately 12.2 mM. A precipitate of the test material was observed at and above 262 µg/mL in all three of the exposure groups. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups.

In conclusion, trimagnesium bis(orthophosphate) 4 -hydrate and thus magnesium hydrogenorthophosphate are considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Justification for classification or non-classification

In accordance with Regulation (EC) No. 1272/2008 (EU CLP) no classification for genetic toxicity is proposed. As all three in vitro studies were negative no further consideration is required for this endpoint.

 

In addition, the Mg2+and PO43-ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.