Reaction mass of diiron carbide, diiron phosphide and triiron phosphide

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997-07-21

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998-08

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guidelilnes S2A

Deviations:

no

Test guideline 5

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of diiron carbide, diiron phosphide and triiron phosphide

EC Number:

701-438-1

Cas Number:

not applicable

Molecular formula:

not applicable (multi constituent substance)

IUPAC Name:

Reaction mass of diiron carbide, diiron phosphide and triiron phosphide

Method

Target gene:

not applicable

Species / strain

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

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.