Ferrocene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro: there are 8 studies included in the dossier; 4 AMES, 2 gene mutation mouse lymphoma assay (MLA) and 2 chromosome abberations. In Vivo; there are 2 studies included the dossier; a mammalian erthrocyte micronucleus tes and a Drosophila SLRL and reciprocal translocation tests. Under the REACh testing strategy the 3 test in vitro test battery is required. Adequate negative AMES data addresses the bacterial gene mutation end point. A recent MLA study conducted in accordance with the relevant OECD guidance and published recommendations, confirms a lack of mammalian gene mutation potential. No acceptable in vitro cytogenicity data has been generated, but the negative data from the in vivo mouse bone marrow micronucleus study is seen as an adequate substitute. Therefore, under the requirements of REACh it is concluded that ferrocene is devoid of any genotoxic potential

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase, tk +/- locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix was prepared immediately prior to dosing by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0

Test concentrations with justification for top dose:

For Experiment 1 the dose range was 29.06 to 930 µg/ml in the absence of S9 and 1.82 to 116.25 µg/ml in the presence of S9. In Experiment 2 the dose range was 0.25 to 12 µg/ml in the absence of S9 and 2 to 80 µg/ml in the presence of S9

Vehicle / solvent:

dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Experiment 1&2 absence of metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Experiment 1&2 presence of metabolic activation

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Remarks on result:

other: strain/cell type: thymidine kinase, tk +/-

Conclusions:

Interpretation of results: negative

It is concluded that dicyclopentadienyl iron did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included four independent treatments at concentration up to 32 μg/ml in the presence (4 hours) of a rat liver metabolic activation system (at 1 and 2% (v/v) final concentration of S9 fraction) and at concentrations of 232.5 µg/ml and 2 μg/mL in the absence (4 and 24 hours, respectively) of metabolic S9. The maximum doses tested were limited by either acceptable reductions in toxicity (as measured by %RTG) or by precipitate (observed by eye) at the end of treatment in the absence or presence of S9, respectively.

Executive summary:

Ferrocene was assessed for its ability to induce gene mutations in an in vivo assay using mouse lymphoma L5178Y cells and conducted according to OECD Guideline 476.

It is concluded that dicyclopentadienyl iron did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included four independent treatments at concentration up to 32 μg/ml in the presence (4 hours) of a rat liver metabolic activation system (at 1 and 2% (v/v) final concentration of S9 fraction) and at concentrations of 232.5 µg/ml and 2 μg/mL in the absence (4 and 24 hours, respectively) of metabolic S9. The maximum doses tested were limited by either acceptable reductions in toxicity (as measured by %RTG) or by precipitate (observed by eye) at the end of treatment in the absence or presence of S9, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Research in the area of genotoxicity has been prolific, both at the fundamental level and also with respect to comparative analysis of the performance and predictivity of individual tests and combination of tests for risk assessment. For an adequate evaluation of the genotoxic potential of a chemical substance, different endpoints (i.e. induction of gene mutations, structural and numerical chromosomal alterations) have to be assessed, as each of these events has been implicated in carcinogenesis and heritable diseases. Traditionally the 3 test in vitro battery has been used, comprising of a bacterial reverse gene mutation test (typically the Ames test), a mammalian forward gene mutation test (typically the MLA) and a chromosome aberration study (as outlined by REACh). Extensive work by Kirklandet al[[i], [ii]] has shown that a test battery consisting of the Ames and in vitro micronucleus test (or chromosomal aberration test) will detect (410/557 = 73.6%) of rodent carcinogens. When the MLA test is added to this test battery an additional 24 carcinogens are detected (434/557 = 77.9%). Whilst a marginal increase in sensitivity[1]is observed the data are not convincing for a number of reasons (which will not be explored further in this opinion). Any furtherin vitromammalian cell tests in the revised test battery would significantly reduce specificity[2] with no substantial gain in sensitivity. As a result of this extensive work the requirements for genotoxicity testing have been revised globally. ICH[3][[iii]] and UK COM[4][[iv]] have recently published updated guidance and EFSA[5][[v]] has published a recent opinion advocating the revised in vitro two test battery which has impacted upon the guidance for plant protection products.

It is important to note that the REACh endpoint specific guidance requirements for chemical safety assessment were published back in 2008 [[vi]], and whilst revisions have taken place these revisions have not affected the recommendations for mutagenicity testing [[vii]].

The published data on ferrocene will therefore be assessed against the current REACh requirements.

Following a review of the available genotoxicity data for ferrocene several published papers concluded that ferrocene showed evidence of gene mutation in the in vitro mammalian gene mutation mouse lymphoma assay. When the data is assessed against the current guideline requirements and the recommendations of Mooreet al., the data is considered unreliable/ uninterpretable, with no definitive conclusion drawn from the published data. Consequently a GLP, OECD guideline MLA study was conducted. This study returned negative results, with the maximum dose tested limited by toxicity at the end of treatment.

Under the REACh testing strategy the 3 test in vitro test battery is required. Adequate negative AMES data addresses the bacterial gene mutation end point. A recent MLA study conducted in accordance with the relevant OECD guidance and published recommendations confirms a lack of mammalian gene mutation potential. No acceptable in vitro cytogenicity data has been generated, the negative data from the in vivo mouse bone marrow micronucleus study is seen as an adequate substitute. Therefore, under the requirements of REACh it is can be concluded that ferrocene is devoid of any genotoxic potential.

 

[1] In the context of genotoxicity testing sensitivity refers to the correct prediction of rodent carcinogens which arein vivogenotoxins

[2] In the context of genotoxicity testing specificity refers to the correct prediction of non-carcinogens

[3] International Conference on Harmonisation of technical requirements for registration of pharmaceuticals for human use

[4] United Kingdom Committee on Mutagenicity of Chemicals in food, consumer products and the environment

[5] European Food Safety Authority

 

[i] Kirkland, D., Aardema, M., Henderson, L. & Muller, L. (2005). Evaluation of the ability of a battery of threein vitrogenotoxicity tests to discriminate rodent carcinogens and non-carcinogens. 1. Sensitivity, specificity and relative predictivity.Mutation Research,584, pp 1-256.

[ii] Kirkland, D., Reeve, L., Gatehouse, D. & Vanparys, P. (2011). A corein vitrogenotoxicity battery comprising the Ames test plus thein vitromicronucleus test is sufficient to detect rodent carcinogens andin vivogenotoxins.Mutation Research,721, pp 27-73

[iii] International Conference on Harmonisation (ICH) of technical requirements for registration of pharmaceuticals for human use. Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1). Step 4. November 2011.

[iv] Committee on Mutagenicity (COM) of Chemicals in food, consumer products and the environment. Guidance on a strategy for genotoxicity testing of chemical substances. 2011.

[v] European Food Safety Authority (EFSA) (2011). Scientific opinion on genotoxicitytesting strategies applicable to food and feed safety assessment. EFSA Journal9(9): 2379.

[vi] ECHA (2008). Guidance on information requirements and chemical safety assessment. Chapter R.7a: Endpoint specific guidance. European Chemicals Agenc, May 2008.

[vii] ECHA (2008). Guidance on information requirements and chemical safety assessment. Chapter R.7.7: Mutagenicity and carcinogenicity. European Chemicals Agency, May 2008.

Justification for selection of genetic toxicity endpoint
Most reliable upto date study available

Justification for classification or non-classification

From the data available, Ferrocene should not be classified as a mutagen for CLP