Orthophosphoric acid

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:

The study was performed between 26 May 2010 and 24 June 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conclusive, done to a valid guideline (OECD TG 471) and was conducted under GLP conditions. No deviations from the test methods were noted.

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

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:

Orthophosphoric acid

EC Number:

231-633-2

EC Name:

Orthophosphoric acid

Cas Number:

7664-38-2

Molecular formula:

H3O4P

IUPAC Name:

phosphoric acid

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): PHOSPHORIC ACID TECHNICAL GRADE
- Analytical purity: 85.01%
- Lot/batch No.: HU10009
- Expiration date of the lot/batch: Not supplied
- Storage condition of test material: Room temperature in the dark
- Other: Description: Clear colourless slightly viscous liquid

Method

Target gene:

Histidine operon for Salmonella
Tryptophan operon 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

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: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully miscible in sterile distilled water at 50 mg/ml in solubility checks performed in house. Sterile distilled water was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

TA100 strain. Used with metabolic activation (S9 mix) only

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

TA1535 strain. Used with metabolic activation (S9 mix) only

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

TA1535 strain. Used with metabolic activation (S9 mix) only.

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA-

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

WP2uvrA- strain. Used with metabolic activation (S9 mix) only

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 hours at 37°C - all plates.
- Pre-incubation period: Only experiment 2 contained a pre-incubation period.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: the frequency of revertant colonies assessed using a Domino colony counter.

The positive and untreated controls were dosed using the standard plate incorporation method described in Section "Mutation Test - Experiment 1".


Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA-), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material formulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Method: Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.

Mutation Test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 µg/plate) but the formulations were pre-incubated.
Method: Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test material formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.


All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Evaluation criteria:

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 (6) 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

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

RESULTS
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 88 80 111 93 98 86 90 93 76 99 74
+ TA100 98 74 102 88 109 92 80 114 97 90 99
- WP2uvrA- 22 27 18 23 24 19 22 25 21 22 22
+ WP2uvrA- 19 32 30 31 22 23 25 24 22 20 23

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable (not presented here).

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 and Table 2 for Experiment 1 and Table 3 and Table 4 for Experiment 2.
A history profile of vehicle and positive control values for 2008 and 2009 is presented in Appendix 2 (attached).
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, 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 strains of bacteria, 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test Results: Experiment 1 – Without Metabolic Activation

 

Test Period		From: 08 June 2010						To: 11 June 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	108 92 89	(96) 10.2#	22 29 25	(25) 3.5	23 32 27	(27) 4.5		22 15 27	(21) 6.0	16 14 10	(13) 3.1
-	50	86 99 115	(100) 14.5	26 27 27	(27) 0.6	33 29 27	(30) 3.1		14 21 14	(16) 4.0	10 15 16	(14) 3.2
-	150	107 111 97	(105) 7.2	20 25 24	(23) 2.6	24 30 24	(26) 3.5		20 18 15	(18) 2.5	15 16 13	(15) 1.5
-	500	98 103 102	(101) 2.6	29 21 23	(24) 4.2	32 26 30	(29) 3.1		20 19 18	(19) 1.0	12 7 15	(11) 4.0
-	1500	103 103 117	(108) 8.1	23 20 18	(20) 2.5	27 31 26	(28) 2.6		16 14 22	(17) 4.2	10 15 10	(12) 2.9
-	5000	111 119 112	(114) 4.4	25 19 22	(22) 3.0	29 27 22	(26) 3.6		13 21 19	(18) 4.2	12 13 11	(12) 1.0
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		499 514 504	(506) 7.6	306 269 231	(269) 37.5	208 189 240	(212) 25.8		145 145 152	(147) 4.0	559 652 385	(532) 135.5

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

 

 

Table 2. Test Results: Experiment 1 – With Metabolic Activation

 

Test Period		From: 08 June 2010						To: 11 June 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	103 103 87	(98) 9.2#	14 14 13	(14) 0.6	26 30 36	(31) 5.0		24 25 31	(27) 3.8	9 14 14	(12) 2.9
+	50	104 110 99	(104) 5.5	13 7 10	(10) 3.0	25 30 31	(29) 3.2		21 18 23	(21) 2.5	10 15 14	(13) 2.6
+	150	119 100 93	(104) 13.5	12 13 14	(13) 1.0	30 34 32	(32) 2.0		23 25 25	(24) 1.2	13 10 7	(10) 3.0
+	500	90 84 92	(89) 4.2	14 14 15	(14) 0.6	30 24 26	(27) 3.1		18 24 29	(24) 5.5	15 11 11	(12) 2.3
+	1500	104 107 100	(104) 3.5	12 9 14	(12) 2.5	35 29 29	(31) 3.5		27 23 33	(28) 5.0	10 15 9	(11) 3.2
+	5000	86 98 84	(89) 7.6	14 14 12	(13) 1.2	30 30 27	(29) 1.7		24 23 32	(26) 4.9	12 9 14	(12) 2.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		405 389 381	(392) 12.2	219 197 229	(215) 16.4	300 323 324	(316) 13.6		202 133 128	(154) 41.4	242 273 216	(244) 28.5

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

 

 

 

Table 3. Test Results: Experiment 2 – Without Metabolic Activation

 

Test Period		From: 21 June 2010						To: 24 June 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	122 107 101	(110) 10.8#	18 9 15	(14) 4.6	30 21 33	(28) 6.2		32 26 20	(26) 6.0	20 19 15	(18) 2.6
-	50	98 122 98	(106) 13.9	15 11 18	(15) 3.5	35 24 42	(34) 9.1		18 20 23	(20) 2.5	9 27 8	(15) 10.7
-	150	84 101 110	(98) 13.2	13 15 8	(12) 3.6	33 25 31	(30) 4.2		27 16 24	(22) 5.7	12 14 18	(15) 3.1
-	500	123 113 122	(119) 5.5	12 8 13	(11) 2.6	25 43 25	(31) 10.4		26 24 35	(28) 5.9	15 10 16	(14) 3.2
-	1500	112 106 97	(105) 7.5	11 13 15	(13) 2.0	33 37 33	(34) 2.3		31 20 23	(25) 5.7	11 14 14	(13) 1.7
-	5000	89 100 101	(97) 6.7	7 8 20	(12) 7.2	35 31 30	(32) 2.6		27 20 21	(23) 3.8	7 9 10	(9) 1.5
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		507 430 484	(474) 39.5	466 276 298	(347) 103.9	317 330 301	(316) 14.5		152 157 132	(147) 13.2	2206 2210 2107	(2174) 58.3

 

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

 

 

Table 4. Test Results: Experiment 2 – With Metabolic Activation

 

Test Period		From: 21 June 2010						To: 24 June 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	102 107 118	(109) 8.2#	16 18 20	(18) 2.0	40 34 43	(39) 4.6		42 27 24	(31) 9.6	20 11 9	(13) 5.9
+	50	99 108 85	(97) 11.6	11 11 13	(12) 1.2	37 37 37	(37) 0.0		26 24 32	(27) 4.2	15 19 10	(15) 4.5
+	150	81 82 95	(86) 7.8	11 9 22	(14) 7.0	44 42 35	(40) 4.7		37 34 22	(31) 7.9	23 13 9	(15) 7.2
+	500	99 108 136	(114) 19.3	19 13 16	(16) 3.0	33 36 35	(35) 1.5		30 23 26	(26) 3.5	13 7 7	(9) 3.5
+	1500	101 76 75	(84) 14.7	14 9 15	(13) 3.2	32 41 34	(36) 4.7		25 25 30	(27) 2.9	10 11 10	(10) 0.6
+	5000	93 77 84	(85) 8.0	14 9 13	(12) 2.6	40 41 41	(41) 0.6		23 31 29	(28) 4.2	9 12 10	(10) 1.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		592 675 673	(647) 47.4	145 132 152	(143) 10.1	299 262 321	(294) 29.8		188 198 246	(211) 31.0	157 166 161	(161) 4.5

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

The test material was considered to be non-mutagenic with and without metabolic activation and under the conditions of this test.

Executive summary:

Introduction.

The method conforms 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, TA100 and Escherichia coli strain WP2uvrA^-were treated with 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 was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (sterile distilled water) 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 no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at 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.