Iron 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:

November 10, 2015 until November 26, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Regulation (EC) No. 440/2008, dated May 31, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Iron orthophosphate

EC Number:

233-149-7

EC Name:

Iron orthophosphate

Cas Number:

10045-86-0

Molecular formula:

FePO4

IUPAC Name:

iron(3+) phosphate

Test material form:

solid

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Aroclor 1254-induced)

Test concentrations with justification for top dose:

31.6, 100, 316, 500, 2000 or 5000 μg

Two preliminary cytotoxicity tests (plate incorporation test, without and with metabolic activation) were carried out in test strain TA100.
5000 μg/plate was chosen as top concentration as no signs of cytotoxicity were noted.

Vehicle / solvent:

- Vehicle used: 0.05 M HCl solution
- Justification for choice of vehicle: The test item was not soluble in any of the solvents recommended, dimethylsulfoxide (DMSO), ethanol, acetone or 0.05 M H2SO4 solution, therefore HCl was used.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

HCl solution

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation: TA1535, TA100

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

HCl solution

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation: TA98

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

HCl solution

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation: TA1537

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

HCl solution

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation: TA102

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

HCl solution

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation: TA98, TA102, TA1537

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-amino-anthracene

Remarks:

with metabolic activation: TA100, TA1535

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
Preincubation Method:
- Preincubation period: 20 minutes at 37°C

In agar (plate incorporation)/ Preincubation method
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS: 3 per concentration and experiment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (colony counter)

Evaluation criteria:

Mutagenicity
The test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient) is observed;
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.

Cytotoxicity
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate without S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate with and without S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate with S9-mix/ without S9-mix (only plate incorporation test)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate in plate incorporation test without S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate in plate incorporation test with S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Yes, test item precipitation was noted in both experiments, each carried out without and with metabolic activation, at concentrations of 316 μg/plate and higher in all test strains.

RANGE-FINDING/SCREENING STUDIES: Yes top concentration was determined to be 5000 μg/plate.


HISTORICAL CONTROL DATA (ranges, means and standard deviation and confidence interval (e.g. 95%) see table no.1)
- Positive historical control data: Valid, within the expected range
- Negative (vehicle) historical control data: Valid, within the expected range

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Colony counter
Cytotoxicity (reduction of the number of revertants by more than 50%) was noted in the following experiments at the top concentration of 5000 μg/plate:

Plate incorporation test:
without S9: in test strains TA98, TA100, TA1535 and TA1537;
with S9: in test strains TA98, TA102 and TA1537;

Preincubation test:
without S9 in test strains: TA1535 and TA1537
with S9 in test strains: TA98 and TA1537.

Any other information on results incl. tables

Table 1: Historical control data of negative and positive control values of the years 2012-2014 (n=97 studies)

Data obtained from plate incorporation and preincubation tests

Negative Reference Item

Strain	TA98		TA100		TA102		TA1535		TA1537
S9 -mix	-	+	-	+	-	+	-	+	-	+
Mean	29.9	31.4	144.0	143.7	275.3	279.3	18.6	18.1	6.5	6.6
SD	6.1	6.3	20.5	20.2	16.0	16.6	4.5	4.5	2.1	2.3
Min	12	18	100	101	224	245	10	8	2	0
Max	49	50	191	189	319	319	31	42	11	18

 

Positive Reference Item

Strain	TA98		TA100		TA102		TA1535		TA1537
S9 -mix	-	+	-	+	-	+	-	+	-	+
	2-NF	BP	SA	2-AA	MMC	BP	SA	2-AA	9-AC	BP
Mean	178.8	176.9	925.2	932.9	972.4	963.8	140.4	142.6	90.9	90.4
SD	65.1	65.1	96.3	88.4	115.8	104.8	44.8	45.6	40.0	38.4
Min	91	83	463	703	759	757	51	49	26	28
Max	434	433	1209	1181	1637	1571	382	371	272	257

 

2-NF : 2-nitro-fluorene

BP: Benzo[a]pyrene

SA: Sodium azide

2-AA: 2-amino-anthracene

MMC: Mitomycin C

9-AC: 9-amino-acridine

 

Main test

Table 1: Plate incorporation test; without metabolic activation

Test substance µg/plate		Number of reverted colonies
		TA98	TA100	TA102	TA1535	TA1537
31.6	mean	34.7	120.0	288.7	19.0.	9.0
	SD	2.1	3.6	11.8	5.3	1.0
100	mean	36.0	119.0	258.3	15.7	9.3
	SD	2.6	2.6	2.9	5.5	0.6
316	mean	30.7 #	123.0 #	284.0 #	16.3 #	8.3 #
	SD	8.7	6.1	2.6	1.2	1.2
500	mean	24.0 #	137.7 #	264.7 #	18.0 #	6.0 #
	SD	3.5	3.2	1.5	1.0	1.0
2000	mean	21.0 #	112.0 #	287.7 #	21.3 #	6.0 #
	SD	1.7	1.0	6.4	4.2	1.7
5000	mean	10.7 #	59.3 #	156.7 #	7.3 #	1.7 #
	SD	0.6	9.9	0.6	3.1	0.6
Negative reference item 100 μL/plate
	mean	27.0	140.3	259.3	18.0	8.3
	SD	1.0	1.2	4.2	4.4	0.6
Positive reference item		2-NF	SA	MMC	SA	9-AC
Concentration μg/plate		10	10	10	10	100
	mean	154.0	966.7	1198.0	136.3	114.7
	SD	11.3	2.5	25.4	10.7	4.0

 

2-NF : 2-nitro-fluorene

SA: Sodium azide

MMC: Mitomycin C

9-AC: 9-amino-acridine

 

# test item precipitation

SD standard deviation

mean (n = 3)

 

Table 2: Plate incorporation test; with metabolic activation

 

Test substance µg/plate		Number of reverted colonies
		TA98	TA100	TA102	TA1535	TA1537
31.6	mean	24.0	121.7	270.3	15.7	5.7
	SD	2.6	15.4	3.8	1.2	1.2
100	mean	24.3	119.0	268.0	18.7	6.0
	SD	4.2	10.4	6.1	6.4	0.0
316	mean	25.3 #	134.7 #	279.7 #	13.7 #	7.3 #
	SD	2.1	8.5	20.2	0.6	2.3
500	mean	37.7 #	113.7 #	261.3 #	14.7 #	6.3 #
	SD	0.6	0.6	11.6	2.5	2.5
2000	mean	34.0 #	122.7 #	293.7 #	13.3 #	5.3 #
	SD	1.0	1.2	4.2	0.6	1.2
5000	mean	11.3 #	70.7 #	128.3 #	7.3 #	1.7 #
	SD	0.6	3.2	0.6	1.2	0.6
Negative reference item 100 μL/plate
	mean	27.7	119.0	274.3	14.0	6.7
	SD	0.6	3.0	18.8	1.0	1.5
Positive reference item		BP	2-AA	BP	2-AA	BP
Concentration μg/plate		10	2	10	2	100
	mean	157.0	963.3	1161.0	135.3	104.7
	SD	8.2	6.0	2.0	15.6	17.2

 

BP: Benzo[a]pyrene

2-AA: 2-amino-anthracene

 

 

Table 3: Preincubation test; without metabolic activation

Test substance µg/plate		Number of reverted colonies
		TA98	TA100	TA102	TA1535	TA1537
31.6	mean	23.0	146.0	264.7	20.3	8.0
	SD	0.0	4.4	1.2	0.6	1.7
100	mean	27.7	144..3	286.3	16.7	5.0
	SD	1.5	7.0	1.5	5.1	1.0
316	mean	30.3 #	148.3 #	272.7 #	18.3 #	7.3 #
	SD	1.2	7.5	11.0	0.6	0.6
500	mean	26.3 #	134.0 #	289.0 #	19.0 #	7.3 #
	SD	4.6	10.0	5.3	3.6	0.6
2000	mean	23.0 #	122.3 #	264.3 #	13.3 #	6.3 #
	SD	2.6	2.1	22.2	2.3	1.5
5000	mean	16.3 #	82.3 #	182.0 #	8.3 #	1.0 #
	SD	3.1	4.0	25.0	0.6	0.0
Negative reference item 100 μL/plate
	mean	26.3	146.3	288.3	19.0	5.0
	SD	5.9	2.9	14.4	7.8	0.0
Positive reference item		2-NF	SA	MMC	SA	9-AC
Concentration μg/plate		10	10	10	10	100
	mean	144.3	916.3	887.3	110.7	52.0
	SD	2.1	10.4	4.7	15.0	1.7

 

2-NF : 2-nitro-fluorene

SA: Sodium azide

MMC: Mitomycin C

9-AC: 9-amino-acridine

 

 

Table 4: Preincubation test; with metabolic activation

Test substance µg/plate		Number of reverted colonies
		TA98	TA100	TA102	TA1535	TA1537
31.6	mean	23.7	146.0	257.3	17.3	3.7
	SD	0.6	9.8	5.5	3.2	0.6
100	mean	27.7	139.3	274.0	17.0	7.7
	SD	1.2	5.0	24.3	7.2	1.2
316	mean	29.0 #	147.3 #	257.3 #	16.3 #	7.0 #
	SD	2.0	3.1	3.5	4.0	2.6
500	mean	27.3 #	149.3 #	275.0 #	23.7 #	7.0 #
	SD	1.2	2.1	10.6	4.7	0.0
2000	mean	30.0 #	141.3 #	275.3 #	19.0 #	8.3 #
	SD	1.7	38.8	4.0	1.0	1.2
5000	mean	17.0 #	69.0 #	191.0 #	7.7 #	1.0 #
	SD	0.0	16.8	2.0	1.5	0.0
Negative reference item 100 μL/plate
	mean	34.7	128.3	281.3	14.7	6.0
	SD	1.2	15.3	27.5	0.6	2.6
Positive reference item		BP	2-AA	BP	2-AA	BP
Concentration μg/plate		10	2	10	2	10
	mean	165.3	910.7	906.7	116.3	56.3
	SD	7.2	5.5	7.6	11.5	15.1

 

BP: Benzo[a]pyrene

2-AA: 2-amino-anthracene

Applicant's summary and conclusion

Conclusions:

The test item was determined to be not mutagenic in the bacterial reverse mutation test with Salmonella typhimurium strains with and without metabolic activation.

Executive summary:

In the GLP and OECD guideline 471 compliant bacterial reverse mutation study (Ames test) the test item was tested for mutagenicity in 5 Salmonella typhimurium strains.

Following strains were used TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

In the Preliminary test ranging from 0.316 to 5000 μg/plate the top concentration for the main study was determined to be 5000 μg/plate, where no signs of cytotoxicity were noted.

In the main study the test item was suspended in 0.05 M HCl solution for concentrations of 316, 500, 2000 or 5000 μg per plate. For concentrations lower than 100 μg/plate, the test item was completely dissolved. The vehicle 0.05 M HCl solution served as the negative control.

Test item precipitation was noted in both experiments, each carried out without and with metabolic activation, at concentrations of 316 μg/plate and higher in all test strains. In addition, cytotoxicity (reduction of the number of revertants by more than 50%) was noted in the following experiments at the top concentration of 5000 μg/plate:

 

Plate incorporation test:

without S9: in test strains TA98, TA100, TA1535 and TA1537;

with S9: in test strains TA98, TA102 and TA1537;

Preincubation:

without S9 in test strains: TA1535 and TA1537

with S9 in test strains: TA98 and TA1537

 

No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to concentration of 5000 μg/plate in any of the 5 test strains in two independent experiments without and with metabolic activation. The positive control items showed a significant increase in the number of colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.