Aluminium orthophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following studies are provided:

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

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

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

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 22 December 2009 and 05 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar 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 insoluble in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in–house. The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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 TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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 TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: at 5µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

with S9 mix Migrated to IUCLID6: 2-Aminoanthracene at 2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene at 10 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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 muation rate for TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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 for TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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:

Sponateous muation rate for TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene at 2 µg/plate

Remarks:

with 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.


OTHER EXAMINATIONS: None

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 10E9 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 µg/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 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material formed the best doseable suspension in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house.
- Precipitation: A pale, fine precipitate was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

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 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	82	105	111	111	114	108	109	126	109	103	120P
+	TA100	104	94	99	50	63	83	69	82	90	77	92P
-	WP2uvrA^-	26	26	20	29	26	32	15	25	19	29	28P
+	WP2uvrA^-	28	32	30	25	31	26	29	27	28	24	25P

P         Precipitate

The test material caused a visible reduction in the growth of the bacterial background lawn of Salmonella strain TA100, without S9-mix, at 5000 µg/plate in the range-finding test only. However, this response was not observed in either the preliminary toxicity test or main test and was, therefore, considered spurious and of no biological relevance. No toxicity was noted to any of the remaining bacterial tester strains at any test material dose level in either the absence or presence of S9-mix. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pale, fine precipitate was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

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 or exposure method.

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:

Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test (Experiment 1)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
93		14		43		13		11
120	(110)	13	(16)	24	(33)	23	(19)	11	(12)
118		22		33		20		14

Main Test (Experiment 2)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
100		20		21		22		5
102	(101)	18	(19)	16	(20)	22	(22)	7	(7)
100		18		24		21		9

Test Results: Range-Finding Test– Without Metabolic Activation

Test Period		From: 17 January 2010					To: 20 January 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	102 95 99	(99) 3.5#	29 31 19	(26) 6.4	36 35 40		(37) 2.6	20 21 13	(18) 4.4	8 13 16	(12) 4.0
-	50	97 72 91	(87) 13.1	26 28 24	(26) 2.0	35 36 37		(36) 1.0	21 16 15	(17) 3.2	19 19 11	(16) 4.6
-	150	80 88 81	(83) 4.4	21 30 32	(28) 5.9	38 38 38		(38) 0.0	19 17 22	(19) 2.5	21 13 14	(16) 4.4
-	500	87 85 91	(88) 3.1	20 24 16	(20) 4.0	33 36 41		(37) 4.0	15 14 10	(13) 2.6	11 16 14	(14) 2.5
-	1500	91 94 90	(92) 2.1	24 21 16	(20) 4.0	37 36 41		(38) 2.6	22 11 20	(18) 5.9	11 15 13	(13) 2.0
-	5000	62 TP 64 TP 83 TP	(70) 11.6	21 P 30 P 12 P	(21) 9.0	31 P 43 P 34 P		(36) 6.2	18 P 21 P 28 P	(22) 5.1	14 P 16 P 14 P	(15) 1.2
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		530 657 665	(617) 75.7	1521 1622 1317	(1487) 155.4	913 956 898		(922) 30.1	153 156 188	(166) 19.4	595 896 1010	(834) 214.4

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

T        Partial absence of bacterial background lawn

#        Standard deviation

Test Results: Range-Finding Test– With Metabolic Activation

Test Period		From: 17 January 2010					To: 20 January 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	77 90 77	(81) 7.5#	18 16 25	(20) 4.7	46 38 47		(44) 4.9	24 29 21	(25) 4.0	19 17 16	(17) 1.5
+	50	90 63 71	(75) 13.9	9 9 12	(10) 1.7	34 41 49		(41) 7.5	21 25 27	(24) 3.1	10 20 12	(14) 5.3
+	150	97 77 C	(87) 14.1	17 20 26	(21) 4.6	38 48 45		(44) 5.1	30 24 21	(25) 4.6	9 15 17	(14) 4.2
+	500	85 68 77	(77) 8.5	18 29 21	(23) 5.7	30 40 41		(37) 6.1	17 27 21	(22) 5.0	15 15 13	(14) 1.2
+	1500	80 83 94	(86) 7.4	9 19 27	(18) 9.0	47 40 36		(41) 5.6	27 28 24	(26) 2.1	12 14 12	(13) 1.2
+	5000	75 P 77 P 76 P	(76) 1.0	22 P 22 P 21 P	(22) 0.6	37 P 47 P 39 P		(41) 5.3	22 P 16 P 27 P	(22) 5.5	16 P 5 P 11 P	(11) 5.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		846 1120 1065	(1010) 144.9	187 231 228	(215) 24.6	247 276 297		(273) 25.1	97 157 152	(135) 33.3	242 290 307	(280) 33.7

BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

C        Contaminated

P        Precipitate

#        Standard deviation

Test Results: Main Test– Without Metabolic Activation

Test Period		From: 02 February 2010					To: 05 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	93 94 94	(94) 0.6#	13 19 18	(17) 3.2	18 17 17		(17) 0.6	20 19 19	(19) 0.6	15 8 8	(10) 4.0
-	15	89 98 98	(95) 5.2	19 18 16	(18) 1.5	19 19 16		(18) 1.7	15 15 16	(15) 0.6	13 15 13	(14) 1.2
-	50	79 80 81	(80) 1.0	22 22 16	(20) 3.5	16 15 15		(15) 0.6	24 15 22	(20) 4.7	12 15 10	(12) 2.5
-	150	83 87 87	(86) 2.3	19 19 20	(19) 0.6	16 16 15		(16) 0.6	22 18 18	(19) 2.3	12 14 12	(13) 1.2
-	500	84 86 84	(85) 1.2	17 15 17	(16) 1.2	20 18 18		(19) 1.2	15 20 20	(18) 2.9	12 12 11	(12) 0.6
-	1500	77 81 76	(78) 2.6	15 15 16	(15) 0.6	18 20 12		(17) 4.2	21 16 22	(20) 3.2	15 14 13	(14) 1.0
-	5000	98 P 100 P 97 P	(98) 1.5	22 P 17 P 15 P	(18) 3.6	19 P 15 P 22 P		(19) 3.5	22 P 22 P 16 P	(20) 3.5	13 P 14 P 9 P	(12) 2.6
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		552 593 439	(528) 79.8	436 460 470	(455) 17.5	645 681 685		(670) 22.0	152 154 149	(152) 2.5	735 691 703	(710) 22.7

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

#        Standard deviation

FOR REST OF RESULTS SEE OVERALL REMARKS, ATTACHMENTS

Conclusions:

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

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 also meets the requirements 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 up to six 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 fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended slightly following the results of the range-finding test and the change in test methodology and was 15 to 5000 μg/plate.

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.

The test material caused a visible reduction in the growth of the bacterial background lawn of Salmonella strain TA100, without S9-mix, at 5000 μg/plate in the range-finding test only. However, this response was not observed in either the preliminary toxicity test or main test and was, therefore, considered spurious and of no biological relevance. No toxicity was noted to any of the remaining bacterial tester strains at any test material dose level in either the absence or presence of S9-mix. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A pale, fine precipitate was observed at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

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.

Reference 2

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 07 January 2010 and 05 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15th September 2009, Date of signature: 26th November 2009

Type of assay:

in vitro mammalian chromosome aberration test

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 suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Experiment 1:
Group Final concentration of Aluminium orthophosphate (µg/ml)
4(20)-hour without S9 0*, 20.0, 40.0, 80.0*, 160.0*, 320.0*, 480.0, MMC 0.4*
4(20)-hour with S9 0*, 20.0, 40.0, 80.0*, 160.0*, 320.0*, 480.0, CP 5*
24-hour without S9 0*, 20.0, 40.0, 80.0*, 160.0*, 320.0*, 480.0, MMC 0.2*
* Dose levels selected for metaphase analysis

MMC = Mitomycin C
CP = Cyclophosphamide

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: MEM
- Justification for choice of solvent/vehicle: MEM was selected as the solvent because the test material was readily soluble in it at the required concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 Migrated to IUCLID6: (CP)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period:
48 hrs

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

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

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

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

SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine

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

NUMBER OF REPLICATIONS:
Duplicate cultures

NUMBER OF CELLS EVALUATED:
100/culture

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

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

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

OTHER:
None.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material suspended in MEM
- Precipitation: SEE PRELIMINARY TOXICITY TEST

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test

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


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


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

MMC = Mitomycin C
CP = Cyclophosphamide

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

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

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Historical Aberration Ranges for Vehicle Control Cultures

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

 

	Pulse exposure –S9		Pulse exposure +S9 (1% S9)		24-Hour –S9		Pulse exposure +S9 (2% S9)
	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids
Minimum	0	0	0	0	0	0	0	0
Maximum	2.5	1	2	0.5	2.5	0.5	2.0	0.5
Mean	0.73	0.06	0.53	0.02	0.53	0.03	0.52	0.05
Standard Deviation	0.71	0.21	0.49	0.09	0.68	0.12	0.57	0.16
Number	32	32	32	32	32	32	28	28

2.2      Historical Aberration Range for Positive Control Cultures

	Pulse exposure –S9 MMC 0.4 µg/ml		Pulse exposure +S9 (1% S9) CP 10 - 12.5 µg/ml		24-Hour –S9 MMC 0.2 - 0.25 µg/ml		Pulse exposure +S9 (2% S9) CP 10 - 12.5 µg/ml
	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids
Minimum	7.5	0	10.0	0	6.5	0	8.0	0
Maximum	62.0	0.7	49.0	1	67.0	1.0	38.0	1.0
Mean	34.79	0.02	25.80	0.12	37.79	0.05	22.38	0.04
Standard Deviation	15.79	0.12	8.56	0.29	16.62	0.20	7.62	0.19
Number	32	32	32	32	32	32	27	27

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

Table 1: Mitotic Index - Preliminary Toxicity Test

Table 2: Mitotic Index - Experiment 1

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

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

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

Table 7:  Mean Frequency of Polyploid Cells (%)

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 in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

The study is considered to be of sufficient adequacy, reliability and relevance to be used as a key study for this endpoint.

Executive summary:

Introduction. 

This report describes the results of anin vitrostudy 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 (Scottet al, 1990). The method used followed that described in the 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 three dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, ie. 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 lymphocytesin vitro.

Reference 3

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

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 (incl. QA statement)

Remarks:

Date of inspection: 15th September 2009, Date of signature: 26th 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:

With no evidence of any marked toxicity in the preliminary toxicity test, the maximum dose level in the mutagenicity test was limited by the presence of excessive precipitate.

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 solvent/vehicle:
Formed a suspension suitable for dosing

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 20 to 320 µg/ml for all three of the exposure groups.

With no evidence of any marked toxicity in the preliminary toxicity test, the maximum dose level in the mutagenicity test was limited by the presence of excessive precipitate. A precipitate of the test material was observed at and above 20 µg/ml at the end of the exposure period in all three of the exposure groups in the mutagenicity test. 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 Interpretation of Results in "Any other information and methods incl. tables" section. As this section will not accommodate the required information.

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:

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:

RESULTS

Preliminary Toxicity Test

The dose range of the test material used in the preliminary toxicity test was 4.88 to 1250 µg/ml.
In all three of the exposure groups there were no marked dose related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. A very modest reduction was observed in the 4-hour exposure group in the absence of metabolic activation. However, with no evidence of any reductions in the 24-hour exposure group in the absence of metabolic activation, or the 4-hour exposure group in the presence of metabolic activation, the reduction observed was considered to be spurious and of no toxicological significance and possibly due to a small amount of the cell pellet being lost with the precipitate during the washing phase. Cloudiness was observed immediately on dosing at and above 78.13 µg/ml in the 4‑hour exposure group in the presence of metabolic activation and the 24‑hour exposure group in the absence of metabolic activation, and at and above 156.25 µg/ml in the 4-hour exposure group in the absence of metabolic activation. Precipitate of the test material was observed immediately on dosing at and above 625 µg/ml in all three of the exposure groups. Therefore, based on the precipitate observations, and also with no evidence of any marked toxicity, it was considered that maximum exposure of the test material to the cells had occurred at approximately 312.5 µg/ml. In the subsequent mutagenicity test, the maximum dose level for all three of the exposure groups was 320 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 no evidence of any marked dose related toxicity following exposure to the test material in either the absence or presence of metabolic activation, as indicated by the %RSGand RTG values (Tables 3 and 6). There was also 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 20 µg/ml at the end of the exposure period.

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

A repeat of the 4-hour exposure with metabolic activation was not performed as per the protocol. The data from a chromosome aberration test in human lymphocytes on this test material (Project Number 2920/0084) was considered to provide adequate justification for its omission as stated in the OECD 476 test guideline.

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 no evidence of any marked dose related toxicity following exposure to the test material, as indicated by the %RSGand RTG values (Table 9). There was also no evidence of any reductions in viability (%V), therefore indicating that no residual toxicity had occurred. The positive control induced acceptable levels of toxicity (Table 9).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had no 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 20 µg/ml at the end of the exposure period.

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

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 20 to 320 µg/ml for all three of the exposure groups.

Results. 

With no evidence of any marked toxicity in the preliminary toxicity test, the maximum dose level in the mutagenicity test was limited by the presence of excessive precipitate. A precipitate of the test material was observed at and above 20 µg/ml at the end of the exposure period in all three of the exposure groups in the mutagenicity test. 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)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

All studies are conducted to the appropriate modern OECD guideline and under the conditions of GLP.

Justification for classification or non-classification

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