Trimanganese bis(orthophosphate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (OECD 471; GLP)

In vitro mammalian cell micronucleus test: negative (OECD 487; GLP)

In vitro mammalian cell gene mutation test: negative (OECD 490; GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-02-25 to 2015-06-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

yes

Remarks:

historical control data incomplete (benzo(a)pyrene data missing; positive control data not clearly labelled); Preliminary results missing; strain charac. confirmations not fully described. Proficiency of the lab not comparable with other laboraotries.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature 15 - 25 °C

Target gene:

TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
E. coli WP2 uvrA: trp-

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (containing 10 % S9)

Test concentrations with justification for top dose:

Main test (preincubation method): 0.313, 0.625, 1.25, 2.5, and 5 mg/plate (with and without metabolic activation)
Confirmatory test (plate incorporation method): 0.313, 0.625, 1.25, 2.5, and 5 mg/plate (with and without metabolic activation)
In preliminary tests the highest concentration was determined considering the criteria solubility and cytotoxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: this medium is not suspected of chemical reaction with the test substance and is compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene / daunomycin

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR 191 acridine

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation (main test) and in agar (plate incorporation, confirmatory test);

MAIN TEST (preincubation with and without metabolic activation)
The test solution (0.1 mL) was preincubated with the test strain (0.1 mL, containing approx. 10E8 viable cells) and sterile buffer or the metabolic activation system (0.5 mL) for 20 minutes at 30 °C - 37 °C prior to mixing with the overlay agar (2.0) mL. Tubes were aerated during the pre-incubation by using a shaker.
The content of each tube was poured over the surface of a minimal agar plate. This overlay agar was allowed to solidify before incubation.
After a period of incubation (about 48 - 72 hours, 37 ± 1 °C) revertant colonies were counted and compared with the number of spontaneous revertants in an untreated and/or solvent control culture.
The bacterial colonies were counted using the photo system "Argus X1/Total Lab-software".

CONFIRMATORY TEST (plate incorporation with and without metabolic activation)
For the test without metabolic activation 0.1 mL of the test solutions, 0.1 mL of fresh bacterial culture and 0.5 mL of sterile buffer were mixed with the overlay agar.
For the assay with metabolic activation 0.5 mL of S9 mixture was mixed with the overlay agar (2.0 mL), together with the bacteria and test solution.
The content of each tube was mixed and poured over the surface of a minimal agar plate.
This overlay agar was allowed to solidify before incubation.
After a period of incubation (about 48 - 72 hours, 37 ± 1 °C) revertant colonies were counted and compared with the number of spontaneous revertants in an untreated and/or solvent control culture.

NUMBER OF REPLICATIONS: triplicate plating was used at each dose level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
A preliminary experiment was conducted to determine cytotoxicity and solubility of the test substance. Starting with the test material concentration of 5 mg/plate and subsequent serial dilutions (ratio 1:2) following concentrations of test material were applied: 5, 2.5, 1.25, 0.625, 0.313, and 0.156 mg/plate on two bacteria strains (TA 98 and TA 1535) with different mutation type (frameshift and base-pair substitution) with and without metabolic activation. The highest concentration of that solution was chosen for the main test that caused low cytotoxicity and / or where the precipitate did not interfere with the scoring according to OECD 471.

Rationale for test conditions:

A preliminary experiment was conducted to determine cytotoxicity and solubility of the test substance. The highest concentration of that solution was chosen for the main test that caused low cytotoxicity and / or where the precipitate did not interfere with the scoring according to OECD 471.

Evaluation criteria:

A 2 or 2.5-fold increase in the number of revertant colonies/plate over the background (spontaneous revertant frequency) is used as a criterion to distinguish active mutagens from non-mutagenic materials (MOLTOX Salmonella Mutagenicity Test Kit Instruction Manual, 2014). The presence of dose response is a further criterion for mutagenic materials.

Statistics:

Means of individual plate counts (triplicates) were calculated for test solutions and controls.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitations were seen in all test material concentrations which did not influeunce the results of the assay.

MAIN TEST (preincubation)
No genotoxic activity caused by the test material was detected under the described test conditions. In addition, a presence of test dose response was not detected.
Please also refer to the field "Attached background material" below.

CONFIRMATORY TEST (plate incorporation)
No genotoxic activity caused by the test material was detected under the described test conditions. In addition, a presence of test dose response was not detected.
Please also refer to the field "Attached background material" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data / Negative (solvent/vehicle) historical control data:
The colony counts of negative and positive controls are in the dimensions of historical data and fulfil the requirements according to the MOLTOX Salmonella Mutagenicity Test Kit Instruction Manual (positive control frequencies are at least 2 times of negative control counts (spontaneous frequency)). The results of negative and positive controls confirm the sensitivity and accuracy of the test system.
Please also refer to tables 1 to 3 in the field "Any other information on results incl. tables" below.

Table 1: Historical data of colonies per plate (negative control preparations without metabolic activation) 

Strain	MIN	MAX	MEAN	SD	N
TA98	13	54	29	16	5
TA100	62	246	112	78	5
TA1535	7	28	14	8	5
TA1537	3	8	5	2	4
WP2uvrA	10	17	14	3	4

Table 2: Historical data of colonies per plate (negative control preparations with metabolic activation)

Strain	MIN	MAX	MEAN	SD	N
TA98	21	65	38	19	5
TA100	63	232	108	70	5
TA1535	8	20	11	5	5
TA1537	3	6	5	1	4
WP2uvrA	10	20	15	5	4

Table 3: Historical data of colonies per plate (positive control preparations)

Strain	Chemical	MIN	MAX	N
TA98	2-Aminoanthracene	135	> 1000	5
	Daunomycin	198	> 500	5
TA100	2-Aminoanthracene	326	> 500	5
	Sodium Azide	274	> 500	5
TA1535	2-Aminoanthracene	67	> 300	5
	Sodium Azide	83	> 300	5
TA1537	2-Aminoanthracene	107	210	4
	ICR 191 Acridine	82	2010	4
WP2uvrA	2-Aminoanthracene	309	407	4
	4-Nitroquinoline-1-oxide	839	1419	4

 

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-10 to 2015-11-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014-09-26

Deviations:

yes

Remarks:

no harvest at the end of treatment period after continuous treatment (harvest was conducted 20 hours after test item removal); number and sex of donors unknown; acceptability criteria not fully described

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2014-05-14

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10°C to +25°C, in a tightly closed container and store in a cool, dry and well-ventilated place.

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human (peripheral)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy human donors (non-smoking, no known recent exposures to genotoxic chemicals or radiation)
- Sex, age and number of blood donors: approx. 18 - 35 years of aga/ male or female individuals
- Whether whole blood or separated lymphocytes were used: small innocula of whole blood (0.5 mL) were added to tubes containing chromosome complete culture medium with phytohemagglutinin and penicillin/streptomycin. The tubes are sealed and incubated at 37 °C, and shaken occasionally to prevent clumping.

Cytokinesis block (if used):

5 µg/mL cytochalasin B (CytoB)(exposure duration: 20 hours)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (components per 15 mL): rat liver S9 (1.5 mL); 0.4 M MgCl2 + 1.65 M KCl salt solution (0.3 mL); glucose-6-phosphate (5.41 mM; 21.1 mg); NADP (3.89 mM;45.9 mg; 20 mM Hepes buffer (5.7 mL; pH 7.4); phosphate buffer (7.5 mL)

Test concentrations with justification for top dose:

4 hour exposure (without metabolic activation): 31.62, 100, and 250 μg/mL medium
24 hour exposure (without metabolic activation): 31.62, 100, and 250 μg/mL medium
4 hour exposure (with metabolic activation): 10.0, 31.62, 100, and 250 μg/mL medium
In a preliminary experiment cytotoxicity was noted starting at concentrations of 100 or 250 μg test item/mL medium (24-hour or 4-hour exposure) in the experiment without and with metabolic activation, respectively. Test item precipitation was noted at the top concentration of 500 μg test item/mL in both experiments. Hence, 250 µg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.05 M HCl solution
- Justification for choice of solvent/vehicle: the test item was not soluble in any of the solvents recommended: aqueous media, dimethylsulfoxide, ethanol, acetone or 0.05 M H2SO4 solution. The test item was completely soluble as 2.5 mg/mL 0.05 M HCl solution by using an ultrasonic bath at 37 °C for 10 minutes for preparing a solution. 5 mg test item per mL 0.05 M HCl solution led to test item precipitation. Hence, 5 mg of the test item were suspended in 1 mL 0.05 M HCl solution. This suspension was diluted 1:10 with culture medium to obtain a final concentration of 500 μg/mL medium.
2.5 mg test item per mL 0.05 M HCl were completely dissolved. This solution was diluted 1:10 with culture medium to obtain a final concentration of 250 μg/mL medium. Fresh preparations of the test item were prepared on the day of the experiment and used for the treatment in all experimental parts.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.05 M HCl solution

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Details on test system and experimental conditions:

1) RANGE-FINDING/SCREENING STUDIES:
In a preliminary experiment without (24 hour exposure) and with metabolic activation (4 hour exposure) concentrations of 10.0, 31.6, 100, 250, and 500 μg test item/mL medium were employed. Based on the outcome of this study the concentrations for the main test were selected.
At least 500 cells/replicate cell culture (one culture per concentration) were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity. The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).

2) MAIN TEST:
Cell treatment and harvest times for the used human lymphocytes line are as follows:
1) 4 and 24 hour exposure (without S9 mix)
- freshly prepared blood was seeded with chromosome complete culture medium with phytohemagglutinin and penicillin/streptomycin.
- after 48 hours the medium was replaced by fresh Ham’s F10 medium with fetal calf serum (FCS).
- vehicle control and test item treatments were added at a volume of 500 μL and the positive control at a volume of 50 μL (to 4.95 mL medium) to obtain the corresponding target concentrations.
- cultures were then incubated for 4 or 24 hours at +37°C.
- afterwards the medium was removed and the cultures were washed twice with Ham’s F10 medium.
- after addition of chromosome medium containing Cytochalasin B the cultures were incubated for further 20 hours at 37°C.

2) 4 hour exposure (with S9 mix)
- freshly prepared blood was seeded with chromosome complete culture medium with phytohemagglutinin and penicillin/streptomycin.
- after 48 hours the medium was removed and replaced by Ham’s F10 medium with FCS and 0.5 mL S9 Mix.
- vehicle control and test item treatments were added at a volume of 500 μL and the positive control at a volume of 50 μL (to 4.95 mL medium and 0.5 mL S9 mix) to obtain the corresponding target concentrations.
- cultures were then incubated for 4 hours at +37°C.
- afterwards the medium was removed and the cultures were washed twice with Ham’s F10 medium.
- After addition of chromosome medium containing Cytochalasin B the cultures were incubated for further 20 hours at 37°C.

- precipitation of the test item was checked using a light microscopy at the beginning and end of treatment.
- cells were sampled at a time equivalent to about 1.5 times the normal cell cycle length either after the beginning or at the end of treatment.

Number of replicates: 2 replicate cultures/test item concentration

Methods of slide preparation and staining technique used:
- each culture was harvested and processed separately.
- after an incubation of 20 hours with Cytochalasin B, the cultures were centrifuged, the supernatant was discarded and the cells resuspended in KCl.
- after incubation, the cell suspensions were centrifuged.
- supernatant was discarded and freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added.
- cells were left in fixative for 30 minutes followed by centrifugation.
- supernatant was removed and discarded, and the cell pellet was resuspended in about fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette.
- two drops of this cell suspension were dropped onto a microscope slide and left to air-dry.
- slides were then stained using 10% Giemsa.

Number of cells evaluated: the micronucleus frequencies were analysed in at least 2000 binucleated cells/concentration (at least 1000 binucleated cells per culture; two cultures per
concentration). Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.

Determination of cytotoxicity:
- Method: mitotic index; cloning efficiency; relative total growth; other:
At least 500 cells/replicate cell culture (two cultures per concentration) were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity. The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).

Other examinations:
- pH values and osmolality measurements: pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment.

Rationale for test conditions:

The concentrations employed were chosen based on the results of a cytotoxicity study. Cytotoxicity was noted starting at concentrations of 100 or 250 μg test item/mL medium (24-hour or 4-hour exposure) in the experiment without and with metabolic activation, respectively. Test item precipitation was noted at the top concentration of 500 μg test item/mL in both experiments. Hence, 250 µg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

Evaluation criteria:

If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing micronuclei, it is classified as a positive result.

Statistics:

The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for continuity according to YATES (Colquhoun, 1971*).

*Reference:
- Colquhoun, D.: Lectures on Biostatistics, Clarendon Press, Oxford (1971).

Key result

Species / strain:

lymphocytes: human (peripheral)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

250 µg test item/mL medium (with and without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

1) RANGE-FINDING/SCREENING STUDIES:
- test item precipitation was noted at the top concentration of 500 μg test item/mL in both experiments.
- cytotoxicity was noted at concentrations of 100 or 250 μg test item/mL medium in the experiment without and with metabolic activation (24-hour or 4-hour exposure, respectively).

2) MAIN STUDY
Test without metabolic activation (4- and 24-hour exposure):
Test item:
- micronucleus frequencies of cultures treated with the concentrations of 31.62, 100 or 250 μg test item/mL medium in the absence of metabolic activation ranged from 6.0 to 7.5 micronucleated cells per 1000 binucleate cells.
- no dose-related increase in micronucleated cells up to the top concentration of 250 μg test item/mL medium.
- frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.
Vehicle control:
- 6.0 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure. The vehicle results were within the historical control ranges.
Positive control:
- micronucleus frequencies were increased to 35.5 or 22.0 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively.

Test with metabolic activation (4-hour exposure)
Test item:
- micronucleus frequencies of cultures treated with the concentrations of 10.0, 31.62 or 100 μg test item/mL medium (4-h exposure) in the presence of metabolic activation ranged from 5.5 to 7.0 micronucleated cells per 1000 binucleate cells.
- no dose-related increase in the micronucleus frequency.
- at the cytotoxic concentration of 250 μg test item/mL medium only 135 binucleate cells of sufficient quality could be evaluated.
- frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.
Vehicle control:
- mean frequency of 6.5 micronucleated cells per 1000 binucleate cells was observed. The vehicle result was within the historical control ranges.
Positive control:
- micronucleus frequency was increased to 34.5 micronucleated cells per 1000 binucleate cells for the 4-hour exposure.

Please refer to the field "Attached background material" below.

- Cytotoxicity:
Cytotoxicity was noted in the experiments without and with metabolic activation at the top concentration of 250 μg test item/mL medium.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant changes in pH of the test item formulations at concentrations of 10 to 500 μg/mL medium were noted.
- Effects of osmolality: no relevant changes in osmolality of the test item formulations at concentrations of 10 to 500 μg/mL medium were noted.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data / Negative (solvent/vehicle) historical control data: please refer to Table 1 in the field "Any other information on results incl. tables" below.

Table 1: Historical background data in vitro micronucleus test in cultured human peripheral lymphocytes (n = 21; background data from 2013 to 2015)

	Micronucleus frequency per 1000 cells
	without metabolic activation		with metabolic activation
	Untreated control	Vehicle control	Untreated control	Vehicle control
mean	6.6	6.3	6.4	6.1
SD	2.9	3.1	2.6	4.2
range	2.0 - 17	2.0 – 18	3.0 – 13	1 – 22
95% Confidence interval	5.9 – 7.3	5.7 – 6.8	5.7 - 7.1	5.1 - 6.7
	Mitomycin C Positive control	colchicine  Positive control	Cyclophosphamide Positive control
mean	46.9	25.9	44.0
SD	35.0	9.5	37.7
range	17 - 137	15 - 63	14 - 158

Conclusions:

The substance tested non-clastogenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-12 to 2015-12-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2015-07-28

Deviations:

yes

Remarks:

incubation time fafter addition of TFT was 11 to 14 days

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2014-05-14

Type of assay:

other: in vitro mammalian cell gene mutation test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10°C to +25°C, in a tightly closed container in a cool, dry and well-ventilated place.

Target gene:

TK gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

clone 3.7.2C

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (American Type Culture Collection), 0801 University Blvd., Manassas, VA 20110-2209, USA

MEDIA USED
- Type and identity of media:
1) growth medium RPMI 1640 with glutamaxTM medium supplemented with Pluronic® F68, gentamycin, amphotericin B and horse serum (10% by volume).
2) treatment medium: growth medium with a reduced horse serum content (5% by volume).
3) plating medium: growth medium with increased horse serum content but without Pluronic® F68.
4) selection medium: growth medium that contains 3 μg/mL of 5-trifluoro-thymidine (TFT).

- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix: rat liver S9 (3.0 mL); 150 mM KCl salt solution (1.5 mL); glucose-6-phosphate (270.0 mg); NADP (37.5 mg); sterile aqua ad iniectabilia (3.0 mL); treatment medium (15 mL)

Test concentrations with justification for top dose:

Experiment 1: 1.95, 3.9, 7.8, 15.6, and 31.3 µg/mL (with and without metabolic activation; 3-hour exposure)
Experiment 2: 1.95, 3.9, 7.8, 15.6, and 31.3 µg/mL (without metabolic activation; 24-hour exposure)
Experiment 2: 1.95, 3.9, 7.8, 15.6, and 31.3 µg/mL (with metabolic activation; 3-hour exposure)
In a preliminary cytotoxicity study cytotoxicity was noted in the absence and in the presence of metabolic activation at concentrations of 31.3 μg/mL and higher. Hence the top dose for this study was selected to be 31.3 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.05 M HCl solution
- Justification for choice of solvent/vehicle: the test item was not soluble in any of the solvents recommended: aqueous media, dimethylsulfoxide, ethanol, acetone or 0.05 M H2SO4 solution. However, the test item was completely soluble as 2.5 mg/mL 0.05 M HCl solution. 5 mg test item/mL 0.05 M HCl solution led to test item precipitation. Hence, 5 mg of the test item were suspended in 1 mL 0.05 M HCl solution. This suspension was diluted with culture medium to obtain a final concentration of 250 μg/mL medium for determination of cytotoxicity in the preliminary test. Test item precipitation was noted at the top concentration of 250 μg test item/mL in both preliminary experiments. Therefore, 250 μg of the test item was the maximum concentration tested in this study limited by solubility. In the main study the test item was completely dissolved to a concentration of 1.25 mg/mL 0.05 M HCl. This solution was then further diluted to the appropriate concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.05 M HCL in culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

1) RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity experiment at concentrations of 7.8, 15.6, 31.3, 62.5, 125 and 250 μg test item/mL medium with and without metabolic S9-activation was performed to establish an appropriate concentration range for the mutation experiment.
For the evaluation of the cytotoxicity in the preliminary experiment the relative plating efficiency RPE1 obtained at the time after treatment is sufficient and was evaluated.

Other examinations:
- pH values and osmolality measurements: pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment.

2) MUTAGENICITY TESTING
Experiment 1:
The assay procedure used is based on that reported by COLE et al. (1990)*.
Cells were obtained from logarithmically growing laboratory stock cultures. The cells were pelleted by centrifugation, the culture medium was removed, and the cells were resuspended in treatment medium that contained 5% heat inactivated horse serum and the corresponding concentration of test substance was added with or without metabolic activation. The final cell density at start of treatment was 0.5 cells/mL. The dosed tubes were closed, mixed and placed on a roller drum at approx. 37°C for an exposure period of 3 hours. Thereafter the cells were washed and resuspended in growth medium.
Cell densities were adjusted to 2 x 10^5/mL and the cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture were placed in two 96 well microtiter plates (192 wells, averaging 1.6 cells/well), incubated for 1 week (plating efficiency step 1) and at the end the number of viable clones was recorded.
The rest of the cells was incubated for 2 days for the expression period. Within the expression period cell densities were determined after incubation for 24 h and adjusted to 2 x 10^5/mL and incubated for another 24 h.
At the end of the expression period a minimum of 4 concentration levels plus positive and negative control were selected for 5-trifluoro-thymidine (TFT) resistance. The selected cultures were diluted to 1 x 10^4 cells/mL and plated for survival (plating efficiency step 2) and TFT resistance in parallel (plating efficiency for TFT resistance). The plating for survival was identical to the above described method (plating efficiency step 1 in 192 wells with average 1.6 cells/well). For the plating for TFT resistance 3 μg/mL TFT (final concentration) were added to the cultures and 0.2 mL of each suspensions placed into four 96-well microtiter plates (384 wells, averaging 2 x 10³ cells/well). The plates were incubated for 11 to 14 days and wells containing clones were identified microscopically and counted.
The mutant frequency was determined and expressed as mutants/10^6 viable cells.
The number of large and small colonies was recorded. Large colonies are defined as ≥ 1/4 and small colonies < 1/4 of the well diameter of 6 mm.
3-Methylcholanthrene only served as positive control item for mutagenicity but not for small/large colony ratios.

Experiment 2:
An exposure time of 3 hours was used for the repeat experiment with metabolic activation and an exposure time of 24 hours was used for the repeat experiment without metabolic activation to also cover long term effects. The experiments were conducted with the same concentrations as in the first experiment.

Number of replicates: single cultures were used for each test item concentration level and reference item.

Determination of cytotoxicity
- Method: relative total growth (RTG) which includes the Relative Suspension Growth (RSG) during the 2 day expression period and the Relative Plating Efficiency (RPE2) obtained at the time of mutant selection.

*Reference:
- COLE, J., M. FOX, R. C. GARNER, D. B. McGREGOR and J. THACKER. Gene mutation assays in cultured mammalian cells. In, 'Basic Mutagenicity Tests': UKEMS recommended procedures, Ed.: D. J. KIRKLAND et al., Cambridge University Press, Chapter 4, pp. 81 - 114 (1990).

Rationale for test conditions:

Based on the results of the preliminary study the test item concentrations were employed in the mutagenicity test. During the preliminary test cytotoxicity was noted in the absence and in the presence of metabolic activation at concentrations of 31.3 μg/mL and higher, test item precipitation at the top concentration of 250 μg test item/mL.

Evaluation criteria:

The interpretation relies on the use of a predefined induced mutant frequency (i.e. increase in mutant frequency above concurrent control), designated as the Global Evaluation Factor (GEF). The GEF (126 x 10^-6) is based on the analysis of the distribution of the negative control mutant frequency data from participating laboratories (M.M. Moore et al., 2006).
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in mutant frequency above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
A test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in mutant frequency, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
In cases when the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result the data is evaluated by expert judgement and/or further investigations.

*Reference:
- MOORE, M.M., HONMA, M., CLEMENTS, J., BOLCSFOLDI, G., BURLINSON, B., CIFONE, M., CLARKE, J., DELONGCHAMP, R., DURWARD, R., FELLOWS, M., GOLLAPUDI, B., HOU, S., JENKINSON, P., LLOYD, M., MAJESKA, J., MYHR, B., O’DONOVAN, M., OMORI, T., RIACH, C., SAN, R., STANKOWSKI, L.F. JR., THAKUR, A.K., VAN GOETHEM, F., WAKURI, S. AND YOSHIMURA, I. (2006). Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: Follow-Up Meeting of the International Workshop on Genotoxicity Tests – Aberdeen, Scotland, 2003 – Assay Acceptance Criteria, Positive Controls, and Data Evaluation, Environ. Mol. Mutagen., 47 (1):1-5.

Statistics:

- Cytotoxicity (mutagenicity testing):
RTG = RSG x RPE2
RTG = Relative Total Growth; RSG = Relative Suspension Growth; RPE2 = Relative Plating Efficiency
RSG [ SG value (treatment)/ mean SG value (control)] x 100
With SG being the suspension growth, the measure of the growth in suspension during treatment and the expression period. SG was calculated as follows:
SG = a x b x c
a = (D0 post treatment cell count/ pre-treatment cell density)
b = (D1 cell count/ cell count set up on D0 post treatment)
c = (D2 cell count/cell count set up on D1)
RPE2 [PE2 value (treatment)/mean PE2 value (control)] x 100
With PE2 being the plating efficiency obtained at the time of mutant selection. The calculations are based on P(0), the proportion of wells in which a colony
has not grown:
PE2 = [-ln P(0)/number of cells plated per well]
Where P(0) = Number of wells with no colony/Total number of wells
The cloning efficiency (CE) is defined as the plating efficiency in percent (CE=PE2 x 100).

- Mutant frequency:
mutant frequency (MF/10^6): (PEm of mutant cells / PE2 of viable cells) x 10^6

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

clone 3.7.2C

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

31.3 μg trimanganese phosphate/mL (with and without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

1) RANGE-FINDING/SCREENING STUDIES:
During the preliminary test cytotoxicity was noted in the absence and in the presence of metabolic activation at concentrations of 31.3 μg/mL and higher. Test item precipitation was observed at the top concentration of 250 μg test item/mL.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant changes in pH of the test item formulations at concentrations of 7.8 to 250 μg/mL medium compared to the controls were noted.
- Effects of osmolality: no relevant changes in osmolality of the test item formulations at concentrations of 7.8 to 250 μg/mL medium compared to the controls were noted.

2) MUTAGENICITY TESTING
- cytotoxicity: cytotoxicity was noted at the top concentration of 31.3 μg trimanganese phosphate/mL in the absence and presence of metabolic activation
The top concentration of 31.3 μg trimanganese phosphate/mL medium in the experiment without metabolic activation (24 hours exposure) was excluded from evaluation because of the cytotoxicity with a relative total growth (RTG) of 4.

- test item treatment: mutation frequencies of the cultures treated with trimanganese phosphate ranged from 53.52 to 169.14 per 10^6 cloneable cells (3 hours exposure) and from 54.43 to 91.82 per 10^6 cloneable cells (24 hours exposure) in the experiments without metabolic activation and from 50.42 to 197.88 per 10^6 cloneable cells (3 hours exposure, first assay) and from 50.14 to 138.97 per 10^6 cloneable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to170 mutants per 10^6 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation. Only the culture treated with the cytotoxic concentration of 31.3 μg trimanganese phosphate/mL marginally exceeded the range of the negative control values and the normal range of 50 to 170 mutants per 10^6 viable cells (197.88 per 10^6 cloneable cells in the first assay with S9 mix). This increase in MF is below the Global Evaluation Factor (GEF) of 126 x 10^-6 and, hence, not considered to be mutagenic.
No change was observed in the ratio of small to large mutant colonies, ranging from 0.40 to 1.53 for trimanganese phosphate-treated cells and from 0.23 to 1.00 for the negative controls.

- negative control: the values of mutation frequencies of the negative controls ranged from 50.99 to 63.82 per 10^6 cloneable cells in the experiments without metabolic activation and from 50.41 to 74.92 per 10^6 cloneable cells in the experiments with metabolic activation (well within the historical data-range).
The calculations of suspension growth of the negative control replicates were in the range between 8 and 32 following 3-hour treatments or between 32 and 180 following 24-hour treatments.

- positive controls: the positive controls methylmethanesulfonate and 3-Methylcholanthrene caused pronounced increases in the mutation frequency ranging from 754.40 to 1075.41 per 10^6 cloneable cells in the case of methylmethanesulfonate and ranging from 591.69 to 618.32 per 10^6 cloneable cells in the case of 3-Methylcholanthrene.
The colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.20 to 1.31 in the case of methylmethanesulfonate.
The mean relative total growth (RTG) for the positive controls was greater than 10%.

Please also refer to the field "Attached background material" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data / Negative (solvent/vehicle) historical control data: please refer to Table 1 and Table 2 in the field "Any other information on results incl. tables" below.

Table 1: Historical data from 2012 to 2014 (n = 22)

Number of mutant colonies per 10^6cells
3h treatment
	Negative control		Negative control without S9 mix: Solvent#				Positive control
	without S9 mix	with S9 mix	without S9 mix		with S9 mix		without S9 mix	with S9 mix
			Aqueous	Organic	Aqueous	Organic	MMS	3-MC
range:	50.2-163.5	48.5-161.54	56.8-160.5	50.2-159.41	52.6-167.32	51.8-169.51	465.1-2724.1	415.7-3510.3
mean value:	80.8	78.1	98.6	79.3	77.0	77.2	1595.3	1313.5
Standard deviation:	27.2	24.5	44.37	23.1	38.5	21.7	585.6	629.1
Number of mutant colonies per 10^6cells
24h treatment
	Negative control		Negative control without S9 mix: Solvent#				Positive control
	without S9 mix		Aqueous		Organic		without S9 mix (MMS)
range:	49.4-161.54		59.0-155.73		49.4-161.54		585.6-2939.4
mean value:	81.1		78.7		81.5		1563.9
Standard deviation:	27.0		45.2		25.9		599.0

# Aqueous solvent (n = 3): Deionised water; organic solvent: DMSO, acetone, ethanol (n = 19)

 MMS = Methylmethanesulfonate 3-MC = 3-Methylcholanthrene

Table 2: Historical data from 2012 to 2014 (n = 22)

Small : large colonies (colony size ratio)
3h treatment
	Negative control		Negative control: Solvent#				Positive control
	without S9 mix	with S9 mix	without S9 mix		with S9 mix		without S9 mix	with S9 mix
			Aqueous	Organic	Aqueous	Organic	MMS	3-MC
range:	0.45-1.30	0.18-2.00	0.52-1.12	0.45-1.30	0.58-1.40	0.35-1.40	1.20-2.91	0.22-1.55
mean value:	0.88	0.92	0.90	0.88	0.95	0.88	2.04	0.81
Standard deviation:	0.21	0.27	0.07	0.22	0.26	0.24	0.37	0.31
24h treatment
	Negative control		Negative control without S9 mix: Solvent#				Positive control
	without S9 mix		Aqueous		Organic		without S9 mix (MMS)
range:	0.37-1.67		0.71-1.12		0.37-1.67		1.25-3.84
mean value:	0.99		0.86		0.99		2.07
Standard deviation:	0.24		0.12		0.28		0.45

# Aqueous solvent (n = 3): Deionised water; organic solvent: DMSO, acetone, ethanol (n = 19)

 MMS = Methylmethanesulfonate 3-MC = 3-Methylcholanthrene

Conclusions:

The substance tested non-mutagenic and non-clastogenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

The substance was not observed to be mutagenic in a reliable bacterial reverse mutation assay (OECD 471), a reliable in vitro mammalian cell micronucleus test (OECD 487), and a reliable in vitro mammalian cell gene mutation test (OECD 490).

Justification for classification or non-classification

Genetic toxicity in vitro

The substance should not be considered to have a mutagenic potential based on a bacterial reverse mutation assay (OECD 471), an in vitro mammalian cell micronucleus test (OECD 487) and an in vitro mammalian cell gene mutation test (OECD 490). The substance does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.