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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Four in vitro studies are presented on structurally related substances as valid read across candidates: 1,1'-Bis(ferrocenyl)octane, CAS Number 501410-94-2, EC Number 479-710-1 (OECD 471) and Iron(2+) dicyclopenta-2,4-dienide (Ferrocene), CAS Number 102-54-5, EC Number 203-039-3 (two studies comparable to OECD 471 and one study according to OECD guideline 476) in order to investigate the mutagenic potential of 1,1"-isopropylideneferrocene, CAS Number 12609 -95 -9, EC Number 235 -719 -0.

In an OECD 471 study (Ames test), 1,1'-Bis(ferrocenyl)octane was found to be non-mutagenic.

In the case of iron (2 +) dicyclopenta-2,4 -dienide (ferrocene), CAS Number 102 -54 -5, EC Number 203 -039 -3, two studies comparable to OECD 471 were conducted and the substance was found to be non-mutagenic.

A MLA study conducted on iron (2 +) dicyclopenta-2,4 -dienide (ferrocene), CAS Number 102 -54 -5, EC Number 203 -039 -3 according to to OECD 476, the substance did not induce mutation at the tk locus of L5178Y mouse lymphoma cells.

Both 1,1'-Bis(ferrocenyl)octane, CAS Number 501410-94-2, EC Number 479-710-1 and iron (2 +) dicyclopenta-2,4 -dienide (ferrocene), CAS Number 102 -54 -5, EC Number 203 -039 -3 are considered to be close enough in structural integrity to 1,1"-isopropylideneferrocene, CAS Number 12609 -95 -9, EC Number 235 -719 -0 for valid read-across to be enacted.

Therefore, 1,1"-isopropylideneferrocene is considered to be non-mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2004 - May 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Read across data is presented from the structurally related substance, 1,1'-Bis(ferrocenyl)octane, CAS Number 501410-94-2, EC Number 479-710-1. This substance bears a close similarity to 1,1''-isopropylidenediferrocene, CAS Number 12609-95-9, EC Number 235-719-0, the distinction being that the alkyl bridging functionality between the two ferrocene moieties is an octyl derivative as opposed to an isopropyl derivative.
Species / strain / cell type:
other: sat TA 98, 100, 1535, 1537; esc WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (Phenobarbital and beta-Naphthoflavone.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 31.62 100.00, 316.20, 1000.00, 2500.00 and 5000 µg/plate
Concentration range in the main test (without metabolic activation): 31.62 100.00, 316.20, 1000.00, 2500.00 and 5000 µg/plate
Vehicle / solvent:
Solvent: Acetone
Species / strain:
other: sat TA 98, 100, 1535, 1537; esc WP2 uvrA
Metabolic activation:
with
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 5000 µg/plate
Species / strain:
other: sat TA 98, 100, 1535, 1537; esc WP2 uvrA
Metabolic activation:
without
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 5000 µg/plate
Additional information on results:
Observations:
After 48 hours incubation microdrops (not precipitate) could be observed as colloidical chemical phenomenon at the concentrations of 5000.00 and 2500.00 µg/plate.
Remarks on result:
other: Preliminary test
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

According to OECD 471, 1,1'-Bis(ferrocenyl)octane is not mutagenic
Executive summary:

The mutagenicity potential of 1,1'-Bis(ferrocenyl)octane, CAS Number 501410-94-2, EC Number 479-710-1was investigated in accordance with the standardised guideline OECD 471 (Ames test).

An OECD 471 (Ames test) study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results was performed on 1,1'-Bis(ferrocenyl)octane, CAS Number 501410-94-2, EC Number 479-710-1.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This substance is considered to be sufficiently close in structural integrity to 1,1''-isopropylidenediferrocene, CAS Number 12609-95-9, EC Number 235-719-0 so as to justify valid read across.

Under the conditions of the study 1,1'-Bis(ferrocenyl)octane was found to be non-mutagenic both with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Start dates 7 and 13 August 1987, 14 January 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Positive controls only included in the third assay. Colonies counted at 4 days (2-3 days is recommended). No strains included capable of identifying oxidising mutagens and cross-linking agents.
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Positive controls only included in the third assay. Colonies counted at 4 days (2-3 days is recommended). No strains included capable of identifying oxidising mutagens and cross-linking agents.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Read across data is presented from the structurally related substance, iron(2+) dicyclopenta-2,4-dienide (ferrocene), CAS Number 102-54-5, EC Number 203-039-3. This substance bears a close similarity to 1,1''-isopropylidenediferrocene, CAS Number 12609-95-9, EC Number 235-719-0. Therefore, it is considered that read-across is valid.
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from Arochlor 1254-induced rat liver; enzyme activity tested with aminoanthracene
Test concentrations with justification for top dose:
10, 50, 100, 250, 500, 1000, 2000, 2500, 5000 µg/plate (plate incorporation)
50, 100, 500 1000, 2000 µg/plate (1st pre-incubation assay)
10, 50, 250, 1000, 2500 µg/plate (2nd pre-incubation assay)
Vehicle / solvent:
DMSO
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: commonly used vehicle which is not toxic to the bacteria at the concentration used
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
ositive controls only included in experiment 3 Migrated to IUCLID6: TA 98; TA 1538
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive controls only included in experiment 3 Migrated to IUCLID6: TA 100; TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Positive controls only included in experiment 3 Migrated to IUCLID6: TA 1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1); preincubation (experiments 2 and 3)

DURATION
- Preincubation period: 30 min
- Exposure duration: 4 days (in agar)
- Selection time (if incubation with a selection agent): 4 days [recommended is 2-3 days]


SELECTION AGENT (mutation assays): deficient histidine in agar, preventing vigorous growth of nonmutants

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: other: backgound lawn

Evaluation criteria:
At least a doubling in revertants, compared to the controls
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 1 mg/plate and above
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Ferrocene (iron-bis-cyclopentadienyl) was determined to be non-mutagenic in a concentration range from 10 to 5000 µg/plate in either the presence or absence of the liver-microsomes-cofactors-mixture (S9 mix). In the concentration range from 50 to 2000 µg/plate, tested and evaluated with pre-incubation, ferrocene was determined both in the presence and absence of the “S9 mix”, likewise as non-mutagenic.

 

These results allow us to conclude that ferrocene does not have genotoxic effects against the Salmonella typhimurium strains used in the tests. With the bacteria, ferrocene led neither to DNA base pair (TA 100 and TA 1535) nor to frame shift mutations (TA 98, TA 1537 and TA 1538).

 

Conclusions:
Interpretation of results (migrated information):
negative

In a reliable study, ferrocene showed no mutagenic potential when tested at up to 5 mg/plate in an in vitro assay for bacteria mutagenicity (Ames test) with five strains of Salmonella typhimurium, both with and without addition of a mammalian metabolic fraction (S9).
Executive summary:

The mutagenic potential ofiron(2+) dicyclopenta-2,4-dienide (ferrocene), CAS Number 102-54-5, EC Number 203-039-3was investigated in a study which was conducted according to EU Method B. 13/14. However, this study lacked the inclusion of strains capable of detecting oxidising mutagens or cross-linking agents.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This substance is considered to be sufficiently close in structural integrity to 1,1''-isopropylidenediferrocene, CAS Number 12609-95-9, EC Number 235-719-0 so as to justify valid read across.

The test substance was used at concentrations of up to 5 mg/plate in a plate incorporation assay and at up to 2.0 or 2.5 mg/plate in two pre-incubation assays with S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, with and without a rat metabolic activation fraction (S9). The plates were scored for revertant colonies after 4 days incubation [not the guideline recommended 2 -3 days]. Controls and vehicle controls were included in all three assays, but positive controls were only included in the final assay.

No evidence of mutagenic activity was apparent with any strain, with or without S9. The positive control substances gave the expected increases in mutant frequency, confirming the sensitivity of the assay.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1993 (no further details)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Read across data is presented from the structurally related substance, iron(2+) dicyclopenta-2,4-dienide (ferrocene), CAS Number 102-54-5, EC Number 203-039-3. This substance bears a close similarity to 1,1''-isopropylidenediferrocene, CAS Number 12609-95-9, EC Number 235-719-0. Therefore, it is considered that read-across is valid.
Target gene:
Histidine locus (reversion to histidine independence)
Species / strain / cell type:
other: S. Typhimurium strains TA100, TA1535, TA97, TA98, TA102, TA104
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from Aroclor 1254-induced rat and hamster liver
Test concentrations with justification for top dose:
0, 100, 333, 1000, 2222 or 4000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (all strains)
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (TA98)
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation. Migrated to IUCLID6: (TA100, TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation. Migrated to IUCLID6: (TA97)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation. Migrated to IUCLID6: (TA102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
Evaluation criteria:
"If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain of bacteria"
Species / strain:
S. typhimurium, other: TA100, TA1535, TA97, TA98, TA102
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
Positive controls validity:
valid
Additional information on results:
An increase in "revertants" was observed in a few cultures, but were not consistant between replicates and only occurred at precipitating concentrations.
Remarks on result:
other: All strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

In a reliable NTP study, ferrocene exhibited no evidence of mutagenic potential when tested at up to 4.0 mg/plate in an in vitro pre-incubation assay for bacterial mutagenicity(Ames test) using five strains of S. typhimurium, with and without metabolic activation.
Executive summary:

The mutagenic potential ofiron(2+) dicyclopenta-2,4-dienide (ferrocene), CAS Number 102-54-5, EC Number 203-039-3 was investigated in an NTP study which was conducted comparable to OECD 471.ording to EU Method B. 13/14.However, this study lacked the inclusion of strains capable of detecting oxidising mutagens or cross-linking agents.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This substance is considered to be sufficiently close in structural integrity to 1,1''-isopropylidenediferrocene, CAS Number 12609-95-9, EC Number 235-719-0 so as to justify valid read across.

The test substance was used at concentrations of up to 4 mg/plate in a pre-incubation assay with S. typhimurium strains TA1535, TA97, TA98, TA100 and TA102, with and without rat or hamster metabolic activation fractions (S9). The highest concentration used was 4 mg/plate since previous assays had shown a high level of precipitation above this concentration. The plates were scored for revertant colonies after 48 h incubation.

No evidence of mutagenic activity was apparent with any strain, with or without S9. The positive control substances gave the expected increases in mutation frequency, confirming the sensitivity of the assay.

Ferrocene exhibited no mutagenic potential when tested at up to 4 mg/plate in an in vitro assay for bacterial mutagenicity using five strains of S. typhimurium, with and without S9. Thus, according to CLP and DSD regulations ferrocene would not be classified as mutagenic under these test conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2013 - March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian cell gene mutation assay
Specific details on test material used for the study:
Read across data is presented from the structurally related substance, iron(2+) dicyclopenta-2,4-dienide (ferrocene), CAS Number 102-54-5, EC Number 203-039-3. This substance bears a close similarity to 1,1''-isopropylidenediferrocene, CAS Number 12609-95-9, EC Number 235-719-0. Therefore, it is considered that read-across is valid.
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), KCI (33 mM) and MgCI2 (8 mM) in RO.
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 Sulphoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Experiment 1 & ", 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
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 +/-
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
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:

The mutagenic potential of iron(2+) dicyclopenta-2,4-dienide (ferrocene), CAS Number 102-54-5, EC Number 203-039-3 was investigated in a study which was conducted according to guideline OECD 476 ( In Vitro Mammalian Cell Gene Mutation Test) using mouse lymphoma L5178Y cells.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This substance is considered to be sufficiently close in structural integrity to 1,1''-isopropylidenediferrocene, CAS Number 12609-95-9, EC Number 235-719-0 so as to justify valid read across.

It is concluded that dicyclopentadienyl iron did not induce mutation at thetklocus 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

Justification for classification or non-classification