Reaction mass of diiron carbide, diiron phosphide and triiron phosphide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria (OECD TG 471, Ames): Negative with and without metabolic activation (S9 mix).
In-vitro chromosome aberration (OECD TG 473): no evidence of an increase in the frequency of structural chromosome aberrations.
In vitro gene mutation study in mammalian cells (OECD TG 476, Mouse Lymphoma Assay): no evidence of mutagenic activity with or without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 April 2011 - 01 June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to official EC and OECD test guidelines, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guidelilnes S2A

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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):

CELLS USED
For lymphocytes:
- Sex, age and number of blood donors: two healthy, non-smoking male donors
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: blood from donors was pooled
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)
- In this laboratory the cell cycle time for human lymphocytes in whole blood culture is approximately 13-14 hours.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: TRPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM glutamine. Aliquots (0.4 mL blood : 4.5 mL medium : 0.1 mL phytohaemagglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37°C in a 5% CO2 atmosphere for approximately 48 hours. The cultures were gently shaken daily to resuspend the cells.

Cytokinesis block (if used):

colcemid (final concentration: 0.1 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix
- The S9 fraction was obtained from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver. The S9 fraction was purchased from a commercial source (batch number 2697) and stored at -80°C or below.
- S9 mix contained: S9 fraction (10% v/v : test 1 and 25% v/v : test 2), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM).

Test concentrations with justification for top dose:

3.25, 8.13, 20.33, 50.82, 127.05, 317.62, 794.04, and 1985.1 µg/mL (standard limit concentration).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Used in the absence of S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Used in the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 hours in PHA
- Exposure duration/duration of treatment: 3 hours and 21 hours
- Harvest time after the end of treatment (sampling/recovery times): 18 hours recovery (3-hour treatment); no recovery (21-hour treatment)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 0.1 μg/mL.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 37°C. After a 10-minute period of incubation at 37°C, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid).The cell pellets were resuspended, then centrifuged at 500 g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Duplicates cultures were used. 100 metaphase figures were examined from each culture.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Chromosome aberrations were scored according to the classification of the ISCN (1985)*. Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. The vernier readings of all aberrant metaphase figures were recorded.
- Determination of polyploidy: Polyploid cells were noted when seen
- Determination of endoreplication: Endoreduplicated cells were noted when seen

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

*References:
- ISCN (1985) An International System for Human Cytogenetic Nomenclature, HARNDEN, D.G. and KLINGER, H. P. (Eds). S. Karger AG, Basel.

Evaluation criteria:

An assay is considered to be acceptable if the negative and positive control values lie withinthe current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
- Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
- The increases exceed the solvent control range of this laboratory, taken at the 99% confidence limit.
- The increases are reproducible between replicate cultures.
- The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
- Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Statistics:

The number of aberrant metaphase cells in each test substance group was compared with the solvent control value using the one-tailed Fisher exact test.
A Cochran-Armitage test for trend was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the
highest concentration group - this process continues until the trend test is no longer significant.
D20s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity data
In the absence of S9 mix following 21 hour continuous treatment, Ferrophosphorus (Fe3P) caused no significant reduction in the mitotic index at 1985.1 μg/mL, compared to the solvent control value. The concentrations selected for metaphase analysis were 3.25, 794.04 and 1985.1 μg/mL.In the presence of S9 mix (5% v/v final concentration) following 3 hour treatment, Ferrophosphorus (Fe3P) caused no significant reduction in the mitotic index at 1985.1 μg/mL, compared to the solvent control value. The concentrations selected for metaphase analysis were 8.13, 794.04 and 1985.1 μg/mL.

Metaphase analysis
In both the absence and the presence of S9 mix, Ferrophosphorus (Fe3P) caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control. All mean values for the solvent control (culture medium), and all Ferrophosphorus (Fe3P) treatment concentrations were within the laboratory historical control range, when taken at the 99% confidence limit. Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Polyploid analysis
No statistically significant increases in polyploid metaphases were observed during metaphase analysis in either test.

Conclusions:

It is concluded that the test substance Ferrophosphorus (Fe3P) has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 July 2011 - 25 July 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in compliance with GLP, and EC, OECD, and other international test guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2A (1996) and Guideline S2B (1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase (TK+/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y mouse lymphoma (3.7.2c) cells (ATCC, VA, USA)
- Suitability of cells: This test system detects a wide range of genetic damage in viable cells capable of forming colonies.

For cell lines:
- Absence of Mycoplasma contamination: Cell stocks are periodically checked for freedom from
mycoplasma contamination.
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix
- S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source (Lot No.: 2698 (Date of preparation: 22 Dec 2010)) and stored at ca -80°C.
- S9 mix contains: S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0. The co-factors were prepared, neutralised with 1N NaOH and filter sterilised before adding to S9 fraction and R0.

Test concentrations with justification for top dose:

Preliminary toxicity test: 3.88, 7.75, 15.51, 31.02, 62.03, 124.06, 248.13, 496.25, 992.5, and 1985 µg/mL (standard limit concentration).
Mutation tests: 62.03, 124.06, 248.13, 496.25, 992.5, and 1985 µg/mL (standard limit concentration).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: Fe3P was found to form a dosable suspension at 3.97 mg/mL in culture medium as part of a previous Huntingdon Life Sciences study (Chromosome Aberration test, Huntingdon Life Sciences, 2011, Study Code FGE0020).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Used in the absence of S9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Used in the presence of S9 Mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate cultures (test substance and positive control) and quadruplicate cultures (vehicle controls)
- Number of independent experiments: A preliminary toxicity test and two main tests comprising three independent mutagenicity assays.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1.2 x 10^7
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours (with S9 mix) and 24 hours (without S9 mix)
- Harvest time after the end of treatment (sampling/recovery times): After selection period.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
3-hour exposure: After the 3-hour exposure, the cells were washed once, resuspended in R10p to nominally 2 x 10^5 cells/mL and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 10^5 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 10^3 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air.

24-hour exposure: Cultures containing 3 x 10^6 cells were treated for 24 hours with test substance, vehicle, or positive control. At the end of the exposure period, the cells were washed once, resuspended in R10p and counted to ascertain treatment growth. The cultures were then diluted to 2 x 10^5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p, the intention being to retain at least 1 x 10^7 cells. Following this, the procedure was the same as in the 3-hour treatment.
- Selection time (if incubation with a selective agent): After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates.
- Selective medium consisted of R10p containing 4 μg/mL trifluorothymidine (TFT).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

Evaluation criteria:

The test agent was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989)* using a one-sided F-test, where p<0.001. Statistics are only reported if the Global Evaluation Factor is exceeded, and this was accompanied by a significant positive linear trend.

References:
- ROBINSON, W.D., GREEN, M.H.L., COLE, J., HEALY, M.J.R., GARNER, R.C., and GATEHOUSE, D. (1989). Statistical evaluation of bacterial/mammalian fluctuation tests. In: KIRKLAND, D. J. (Ed). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part 111. Statistical Evaluation of Mutagenicity Test Data, p.102-140. Cambridge University Press, Cambridge.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test
- Precipitate (observed by eye at the end of treatment) was observed at a concentration of 1985 μg/mL in both the absence and presence of S9 mix, following a 3 hour exposure.
- Exposure to Ferrophosphorus (Fe3P) at concentrations from 3.88 to 1985 μg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values from 127 to 63% and from 134 to 74% respectively.
- Following a continuous exposure for 24 hours, precipitation (assessed by eye at the end of treatment) was observed at a concentration of 1985 μg/mL. Exposure to concentrations from 3.88 to 1985 μg/mL resulted in RSG values from 101 to 53%.
- Concentrations used in the main test were based upon these data.

Main mutation test 1 - 3 hour treatment in the absence of S9 mix
- Precipitation (assessed by eye at the end of treatment) was observed at a concentration of 1985 μg/mL.
- Relative total growth (RTG) values from 126 to 84% were obtained relative to the vehicle control.
- There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the historical vehicle control values and there were no clear increases in the mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05).
- The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main mutation test 1 - 3 hour treatment in the presence of S9 mix
- Precipitation (assessed by eye at the end of treatment) was observed at a concentration of 1985 μg/mL.
- RTG values from 99 to 76% were obtained relative to the vehicle control.
- There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the levels of toxicity. All mean mutant frequencies of the test concentrations were within the historical vehicle control values and there were no clear increases in the mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05).
- The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.















TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuation greater than 1 unit relative to the solvent control was seen at the highest test concentration.
- Effects of osmolality: No fluctuation greater than 50 mOsm/kg seen in the highest concentration tested, relative to the solvent control.
- Precipitation: Precipitation was seen at 1985 µg/mL, the highest concentration tested.

Conclusions:

It was concluded that Ferrophosphorus (Fe3P) did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 2010 - 07 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to official EC and OECD test guidelines, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Genes of the his and trp operons

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix
- S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source (Lot No.: 2528 (Date of preparation: 23 December 2009)) and stored at approximately -80°C.
- The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water.

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500, and 5000 µg/plate (standard limit concentration).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water containing 0.15% Agar as a suspending agent.

- Justification for choice of solvent/vehicle: Fe3P was found to be insoluble in water, DMSO, ethanol, acetone, and DMF (common solvents compatible with the test system)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Used for TA100 and TA1535 in the absence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Used for TA1537 in the absence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Used for TA98 in the absence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Used for WP2 uvrA (pKM101) in the absence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Used for TA98 and TA1537 in the presence of S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate cultures
- Number of independent experiments: two independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): at least 10^9 per mL
- The test substance was added using the plate incorporation and pre-incubation protocol

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes
- Exposure duration/duration of treatment: ca. 72 hours
- Harvest time after the end of treatment (sampling/recovery times): The revertant colony number was enumerated directly after the exposure period using an automated colony counter (Perceptive Instruments.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.

If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above)
are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.

Statistics:

No statistical analysis was performed on this study.

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Contamination Test
The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.

Assay Validity
- The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.
- The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

First Test
- No evidence of toxicity was obtained following exposure to Ferrophosphorus (Fe3P). A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.
-No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Ferrophosphorus (Fe3P) at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Second Test
No evidence of toxicity was obtained following exposure to Ferrophosphorus (Fe3P). No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Ferrophosphorus (Fe3P) at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Conclusions:

It is concluded that Ferrophosphorus (Fe3P) showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Genetic toxicity in vivo

Description of key information

No in vivo genetic toxicity data available.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A bacterial reverse mutation (Ames) study (Huntingdon Life Sciences, 2011) was conducted to assess the potential for Fe₃P to cause gene mutation. The study was conducted according to OECD test guideline 471, EC guideline method B13/14, and US EPA and Japanese guidelines, and in compliance with GLP.

Four mutant strains of Salmonella Typhimurium (TA1535, TA1537, TA98, and TA100) and one strain of Escherichia coli (WP2 uvrA) were treated with Fe₃P at a series of concentrations (5 µg/plate – 5000 µg/plate); the tests were run with and without metabolic activation (S9 mix).

No increase in the number of revertant colonies was seen relative to the concurrent vehicle controls in any of the strains tested at any concentration, either with or without metabolic activation.

It was concluded that Ferrophophorus, Fe₃P, had showed no evidence of mutagenic activity in this bacterial system under the conditions employed.

 

A chromosomal aberration study (Huntingdon Life Sciences, 2011) was conducted to assess the cytogenetic potential of Ferrophosphorous, Fe₃P, in human lymphocytes. The study was conducted according to OECD test guideline 473, EU guideline B10, and US EPA, Japanese, and ICH guidelines, and in compliance with GLP.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Solvent and positive control cultures were also included. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

No statistically significant increase in the proportion of metaphase cells containing chromosomal aberrations when compared to the concurrent negative controls. It was concluded that Fe₃P had not shown any evidence of causing an increase in the frequency of structural chromosome aberrations in the in vitro cytogenetic test system, under the experimental conditions.

 

A mouse lymphoma assay (Huntingdon Life Sciences, 2011) was conducted to assess the mutagenic potential of Fe₃P. The study was conducted according to OECD guideline 476, EU guideline B17, and US EPA, Japanese, and ICH guidelines, and in compliance with GLP.

Mouse lymphoma L5178Y cells were treated with a suspension of Fe₃P at concentrations up to the typical limit dose of 10 mM (1985 µg/mL), with and without metabolic activation (S9 mix).

No increase in mean mutant frequencies were seen in any of the tests conducted, nor was any reduction in relative total growth (RTG) observed. It was concluded that Fe₃P had not shown any mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Justification for classification or non-classification

In vitro gene mutation in bacteria (OECD TG 471, Ames): Negative with and without metabolic activation (S9 mix).
In-vitro chromosome aberration (OECD TG 473): no evidence of an increase in the frequency of structural chromosome aberrations.
In vitro gene mutation study in mammalian cells (OECD TG 476, Mouse Lymphoma Assay): no evidence of mutagenic activity with or without metabolic activation.

Three in-vitro studies (as described above) examined aspects of the mutagenic potential of Fe₃P. None of these studies indicated any mutagenic potential. In vivo data is not available. Based on this information, it is not necessary to classify Fe₃P as hazardous on the basis of mutagenic activity according to either the CLP Regulation (Regulation (EC) 1272/2008).