Trimagnesium bis(orthophosphate)

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trimagnesium phosphate-4-hydrate

IUPAC Name:

Trimagnesium phosphate-4-hydrate

Test material form:

solid

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

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 2

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 mix

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

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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

Controls 6

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

Controls 7

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

Controls 8

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

Controls 9

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

Controls 10

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

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

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.