Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key studies are submitted for the following REACH endpoints:
- in vitro gene mutation in bacteria (Section 8.4.1)
- in vitro cytogenicity study in mammalian cells (Section 8.4.2)
- in vitro gene mutation study in mammalian cells (Section 8.4.3)

These studies are conducted to the appropriate modern OECD guideline and under the conditions of GLP. Studies have been conducted on the hydrated form of the substance to be registered and therefore in accordance with 'Data Submission Manual, Part 18 - How to report substance identity in IUCLID 5 for registration under REACH' the hydrated form is considered to be different to the anhydrous form. Read-across from trimagnesium phosphate-4-hydrate to the anhydrous trimagnesium bis(orthophosphate) is justified on the basis that the addition of water is unlikely to detrimentally affect the genotoxic profile of the substance.

Studies on analagous substances are considered to be sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation EC (No.) 1272/2008 (EU CLP).

Endpoint Conclusion:No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 22 December 2009 and 07 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category


(1) Source and target substances all inorganic salts of a divalent cation from Group 2 of the periodic table, magnesium,and phosphoric acid. Thus, they share the Mg2+ cation and the PO43- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Mg2+ cations and the PO43- anion.
(3) Genetic toxicity is assessed on the basis of the Mg2+ cation on the basis that the phosphate anion is not considered to be toxicologically relevant - source substance has the highest content of Mg on a %w/w basis.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 15 September 2009, Date of Signature on GLP certificate: 26 November 2009
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation Rate for TA100
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA100
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: at 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: at 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation rate of TA1537
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 2 µg/plate
Remarks:
with S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl suphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix Migrated to IUCLID6: at 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation rate of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix Migrated to IUCLID6: at 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation rate of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix Migrated to IUCLID6: at 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation rate of WP2uvrA-
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 10 µg/plate
Remarks:
with S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation rate of WP2uvrA-
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: at 2 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation
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
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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:
The test material was considered to be non-mutagenic under the conditions of this 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:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

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 dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

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.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 7 January 2010 and 20 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category


(1) Source and target substances all inorganic salts of a divalent cation from Group 2 of the periodic table, magnesium,and phosphoric acid. Thus, they share the Mg2+ cation and the PO43- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Mg2+ cations and the PO43- anion.
(3) Genetic toxicity is assessed on the basis of the Mg2+ cation on the basis that the phosphate anion is not considered to be toxicologically relevant - source substance has the highest content of Mg on a %w/w basis.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
See discussion (deviation does not effect the reliability of the study)
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 15th September 2009, Date of Signature on GLP certificate: 26th November 2009
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
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 suitabilityThe 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 (preliminary test): 13.0 to 3340 μg/mL

Experiment 2 (main test):
4(20)-hour without S9 0*, 52.2, 104.4*, 208.8, 417.5, 835*, 1670*, MMC 0.4*
4(20)-hour with S9 0*, 52.2, 104.4*, 208.8, 417.5, 835*, 1670*, CP 5*
24-hour without S9 0*, 52.2, 104.4*, 208.8, 417.5*, 835, 1670*, MMC 0.2*

* dose levels selected for metaphase analysis
MMC: mytomycin C
CP: cyclophosphamide
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: MEM
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:
Experiment1: 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.
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.

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

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:
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. The test material 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:

SUMMARY

Introduction.

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods.

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.

Results.

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.

Conclusion.

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 18 January 2010 and 08 February 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category


(1) Source and target substances all inorganic salts of a divalent cation from Group 2 of the periodic table, magnesium,and phosphoric acid. Thus, they share the Mg2+ cation and the PO43- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Mg2+ cations and the PO43- anion.
(3) Genetic toxicity is assessed on the basis of the Mg2+ cation on the basis that the phosphate anion is not considered to be toxicologically relevant - source substance has the highest content of Mg on a %w/w basis.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Remarks:
Date of GLP inspection: 15 September 2009 Date of GLP signature: 26 November 2009
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
The maximum dose level used was equivalent to approximately 12.2 mM.

Vehicle and positive controls were used in parallel with the test material. Solvent (R0 medium) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 142314732109252 at 400 µg/ml and 150 µg/ml for the 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
Vehicle used:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.

Justification for choice of vehicle:
Formed a suspension suitable for dosing at the required concentration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

One main experiment was performed. In this main experiment, 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.
Evaluation criteria:
Please see "Any other information on materials and methods incl. tables" section.
Statistics:
Please see "Any other information on materials and methods incl. tables" section.
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 % RSGand 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.
Remarks on result:
other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Remarks:
Migrated from field 'Test system'.

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:
The test material did 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:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Methods. 

One main experiment was performed. In this main experiment, 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.

Results. 

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.

Conclusion. 

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

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

Additional information

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.