Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For mutagenicity read-across data is presented from studies on substances structurally related to 1,1"-isopropylidenebis(ethylferrocene), CAS Number 37206 -42 -1, EC Number 310 -202 -3, those substances being :-

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.

As regards iron(2 +)dicyclopenta-2,4 -dienide (ferrocene), CAS Number 102 -54 -5, EC Number 203 -039 -3 there are 8 in vitro studies available; 4 AMES, 2 gene mutation mouse lymphoma assay (MLA) and 2 chromosome abberations.

In addition there are two in vivo studies available; a mammalian erthrocyte micronucleus test 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.

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

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

Both read across substances on which mutagenicity dats is available are considered to be sufficiently close in structural integrity to 1,1"-isopropylidenebis(ethylferrocene), CAS Number 37206 -42 -1, EC Number 310 -202 -3 so as to justify valid read across.

Therefore, it is consdiered that 1,1"-isopropylidenebis(ethylferrocene), CAS Number 37206 -42 -1, EC Number 310 -202 -3 is also non mutagenic.

Link to relevant study records

Referenceopen allclose all

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:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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:
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
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:

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:
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)
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 structural similarity to 1,1"-isopropylidenebis(ethylferrocene), CAS Number 37206 -42 -1, EC Number 310 -202 -3, the distinction being that the alkyl bridging functionality between the two ferrocene moieties is an octyl derivative as opposed to an isopropyl derivative and that two cyclopentadienyl moieties in the target substance bear ethyl substituents.
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"-isopropylidenebis(ethylferrocene), CAS Number 37206 -42 -1, EC Number 310 -202 -3 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:
key study
Study period:
1980 [no further details given]
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable study performed according to National Toxicology Program protocols. The study did not include strains to detect oxidative mutagens or cross-linking agents as recommended in current OECD guidelines.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Lacking strains to detect oxidising mutagens or cross-linking agents
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus (reversion to histidine independence)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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, 333.3, 1000, 3333.3 or 10,000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 95% ethanol
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:
9-aminoacridine
Remarks:
without metabolic activation

Migrated to IUCLID6: TA1537
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): deficiency in histidine to inhibit growth of non-mutants

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: background lawn


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"
Statistics:
None
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
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 10.0 mg/plate in an in vitro pre-incubation assay for bacterial mutagenicity (ames test) using four strains of S. typhimurium, with and without metabolic activation.
Executive summary:

Ferrocene was assessed for its ability to induce mutation in an in vitro assay for bacterial mutagenicity (Ames test) in a NTP study following a protocol similar to OECD guideline 471, but lacking the inclusion of strains to detect oxidising mutagens or cross-linking agents.

The test substance was used at concentrations of up to 10.0 mg/plate in a pre-incubation assay with S. typhimurium strains TA1535, TA1537, TA98 and TA100, with and without rat or hamster metabolic activation fractions (S9). 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 10.0 mg/plate in an in vitro assay for bacterial mutagenicity using four 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 bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980 [no further details given]
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Reliable study performed according to National Toxicology Program protocols. The study only tested the substance up 100 ug/plate (instead of the recommended 5 mg/plate) and did not include strains to detect oxidative mutagens or cross-linking agents as recommended in current OECD guidelines.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only tested at up to 100 ug/plate, instead of the recommended 5 mg/plate. Lacking strains to detect oxidising mutagens or cross-linking agents
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus (reversion to histidine independence)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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, 0.33, 1.0, 3.3, 10, 33, 66, or 100 ug/plate (TA100)
0, 0.33, 1.0, 3.3, 10, 33 ug/plate (TA1535, TA1537, TA98)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoantracene
Remarks:
all strains 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
Remarks:
without metabolic activation (TA98)
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 (TA1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation (TA100, TA1535)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (TA100 only); preincubation (all strains)

DURATION
- Preincubation period: 20 min.
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): deficiency in histidine to inhibit growth of nonmutants

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: background lawn
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"
Statistics:
None
Species / strain:
other: TA1535, TA1537, TA98
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
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
tested with hamster S9
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
tested with rat S9
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:
In the pre-incubation assay with TA100 two of the replicates gave equivocal results as did the plate incorporation assay, showing a dose-related increase, but which did not reach a doubling of revertant colonies. The other replicate in the pre-incubation assay gave a negative result.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. TA 100 (without metabolic activation)

Dose (µg/plate)

No activation

No activation

No activation

Protocol:

Pre-incubation

Pre-incubation

Pre-incubation

0

70

106

116

0.33

72

111

 

1.0

53

99

 

3.3

65

107

 

10

64

105

116

16

 

 

117

33

77

105

123

66

 

 

128

100

 

 

111

Results:

Negative

Negative

Negative

Table 2. TA 100 (with hamster liver metabolic activation)

Dose (µg/plate)

Hamster S9

Hamster S9

Hamster S9

Hamster S9

Protocol:

Pre-incubation

Pre-incubation

Plate incorporation

Pre-incubation

0

143

151

136

155

0.33

131

158

 

 

1.0

119

153

 

 

3.3

141

155

 

 

10

137

147

132

153

16

 

 

127

169

33

119

150

136

173

66

 

 

131

153

100

 

 

140

153

Results:

Negative

Negative

Negative

Negative

Table 3. TA 100 (with rat liver metabolic activation)

Dose (µg/plate)

Rat S9

Rat S9

Rat S9

Rat S9

Protocol:

Pre-incubation

Pre-incubation

Plate incorporation

Pre-incubation

0

101

144

123

148

0.33

105

148

 

 

1.0

116

167

 

 

3.3

137

181

 

 

10

134

177

146

132

16

 

 

147

131

33

134

172

156

146

66

 

 

134

126

100

 

 

147

115

Results:

Equivocal

Equivocal

Equivocal

Negative

Conclusions:
Interpretation of results (migrated information):
ambiguous with metabolic activation TA100 with rat S9
negative with metabolic activation TA100 with hamster S9
negative with metabolic activation TA1535, TA1538, TA98
negative without metabolic activation TA1535, TA1537, TA98, TA100

In a reliable NTP study, ferrocene exhibited equivocal evidence of mutagenic potential in S. typhimurium strain TA100 with rat liver metabolic activiation (S9) when tested at up to 100 ug/plate in an in vitro assay (Ames test) for bacterial mutagenesis. No evidence of mutagenic activity was observed in strains TA1535, TA1537 or TA98, with and without S9, or in TA100 (without S9 or with hamster S9).
Executive summary:

Ferrocene was assessed for its ability to induce mutations in an in vitro assay for bacterial mutagenicity (Ames test) in a NTP study following a protocol similar to OECD guideline 471, but at up to 33 and 100 ug/plate only (instead of the recommended 5 mg/plate) and lacking the inclusion of strains to detect oxidising mutagens or cross-linking agents.

The test substance was used at concentrations of up to 100 ug/plate in a plate incorporation and pre-incubation assay with S. typhimurium strain TA100 and at up to 33 ug/plate with strains TA1535, TA1537 and TA98 in a plate incorporation assay. All strains were tested without, and with rat and hamster metabolic activation fractions (S9). The plates were scored for revertant colonies after 48 h incubation.

No evidence of mutagenic activity was apparent with TA1535, TA1537 or TA98, with or without S9, or in TA100, without or with hamster S9. However, there was equivocal evidence of mutagenic activity with TA100, with rat S9 in two of the three replicates in the the pre-incubation assay and the plate incorporation assay. A dose-related increase in revertant colonies was observed, but which did not reach a doubling in number compared to the solvent control. The positive control substances gave the expected increases in mutation frequency, confirming the sensitivity of the assay.

Ferrocene exhibited equivocal mutagenic potential in S. typhimurium strain TA100 with rat S9 but was negative with hamster S9 when tested at up to 100 ug/plate in an in vitro assay for bacterial mutagenicity. However, it showed no mutagenic activity with hamster S9. Three other strains were negative, both with and without S9. Thus, according to CLP and DSD regulations ferrocene would not be classified as mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start dates 7 and 13 August 1987, 14 January 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study with acceptable restrictions. Conducted according to EU Method B.13/14, but without the inclusion of strains to detect oxidising mutagens or cross-linking agents. Colonies counted after 4 days (2-3 days is recommended) and positive controls were only included in the third assay
Qualifier:
according 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:
according 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
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:
Positive 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
Statistics:
none
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'.
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:

Ferrocene was assessed for its ability to induce mutations in an in vitro bacterial reverse mutation assay (Ames test), 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 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.

Ferrocene exhibited no mutagenic potential when tested at up to 5 mg/plate in an in vitro assay for bacterial mutagenicity using five strains of S. typhimurium, with and without S9. Thus, according to EU CLP and DSD regulations ferrocene would not be classified as mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993 [no further details]
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable study performed according to National Toxicology Program protocols.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus (reversion to histidine independence)
Species / strain / cell type:
other: S. typhimurium TA100, TA1535, TA97, TA98, TA102
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
- Exposure duration: 48 h (in plates)

SELECTION AGENT (mutation assays): deficiency in histidine to inhibit growth of nonmutants



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"
Statistics:
none
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:

Ferrocene was assessed for its ability to induce mutations in an in vitro assay for bacterial mutagenicity (Ames test) in a NTP study following a protocol similar to current OECD guideline 471.

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 cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981 (Study started 25 March, no further details given)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable study performed to National Toxicology Program protocols. The study included only a 2 h exposure period with S9 and not the 3-6 h as recommended in current OECD guidelines
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
(only exposed to ferrocene for 2 h with S9, instead of the recommended 3-6 h)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
none (chromosomal mutations)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium supplemented with 10% fetal calf serum and L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
0, 40, 130 and 400 ug/ml with and without S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Justification for choice of solvent/vehicle: Acceptable solvent which is not cytotoxic to the cells at the level used
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
triethylenemelamine
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and experimental conditions:
- Preincubation period: 24 h
- Exposure duration: 2 h without S9, 19 or 22.5 h with S9
- Expression time (cells in growth medium): 17 or 20.5 h (without S9); 19 or 22.5 h (with S9)
- Selection time (if incubation with a selection agent): 2 h after addition of spindle inhibitor
- Fixation time (start of exposure up to fixation or harvest of cells): 19 or 22.5 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1 each for experiments 1 and 2

NUMBER OF CELLS EVALUATED: 100/dose/experiment

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
Evaluation criteria:
An increase in aberrations (excluding gaps) above those seen in the solvent controls
Statistics:
Trend test using a binomial sampling model (percent cells in each category of aberration that was regressed linearly on log-dose, and the test statistic reflected the binomial sampling assumption).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: CHO-W-B1
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

In a reliable NTP study, ferrocene showed no evidence of inducing chromosome aberrations in Chinese hamster ovary cells when tested at up to 400 ug/ml, with and without metabolic activation.
Executive summary:

Ferrocene was assessed for its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells in an NTP study conducted to a protocol similar to OECD guideline 473, but with a shorter exposure period for cultures without metabolic activation (2 h rather than the recommended 3-6 h).

The test substance was used at concentrations of up to 400 ug/ml in two separate experiments, conducted using NTP protocols. Actively growing cultures were exposed for 2 or 19-22.5 h, without and with S9, respectively. Cultures exposed without S9 were washed before completing the 19-22.5 h incubation in fresh medium. Colcemid was added to all cultures two hours before the end of the incubation period to arrest the cells in metaphase. Vehicle control and positive controls were similarly treated. Cells were then spread on slides, fixed, stained with Giemsa and 100 cells per culture were scored for chromosome aberrations.

No increases in chromosome aberrations occurred, either with or without S9. The positive controls gave the expected increases in the frequency of aberrations, confirming the sensitivity of the assay.

Ferrocene showed no evidence of inducing chromosome aberrations in Chinese hamster ovary cells when tested at up to 400 ug/ml, with and without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981 (started 18 May or 29.December, no further details)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable study performed to National Toxicology Program protocols. The study included only a 2 h exposure period with S9 and not the 3-6 h as recommended in current OECD guidelines
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
only exposed to ferrocene for 2 h with S9, instead of the recommended 3-6 h
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
none (chromosomal mutations)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium supplemented with 10% fetal calf serum and L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Prepared from Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
Without S9: experiment 1: 0, 33, 10 or 330 ug/ml; experiment 2: 0, 50, 301 or 402.5 ug/ml

With S9: experiment 1: 0, 33, 10 or 330 ug/ml; experiment 2: 0, 301 or 402.5 ug/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Acceptable solvent which is not cytotoxic to the cells at the level used
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
triethylenemelamine
Remarks:
without S9 (experiment 1)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 (experiment 2)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 (experiments 1 and 2)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: 2 h without S9, 19 or 22.5 h with S9
- Expression time (cells in growth medium): 17 or 20.5 h (without S9); 19 or 22.5 h (with S9)
- Selection time (if incubation with a selection agent): 2 h after addition of spindle inhibitor
- Fixation time (start of exposure up to fixation or harvest of cells): 19 or 22.5 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1 each for experiments 1 and 2

NUMBER OF CELLS EVALUATED: 100/dose/experiment

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no


Evaluation criteria:
An increase in aberrations (excluding gaps) above those seen in the solvent controls
Statistics:
Trend test using a binomial sampling model (percent cells in each category of aberration that was regressed linearly on log-dose, and the test statistic reflected the binomial sampling assumption).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: CHO-W-B1
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

In a reliable NTP study, ferrocene showed no evidence of inducing chromosome aberrations in Chinese hamster ovary cells when tested at up to 402.5 ug/ml, with and without metabolic activation.
Executive summary:

Ferrocene was assessed for its ability to induce chromosome aberrations in Chinese hamster ovary cells in an NTP study conducted to a protocol similar to OECD guideline 473, but with a shorter exposure period for cultures without metabolic activation (2 h rather than the recommended 3 -6 h).

The test substance was used at concentrations of up to 402.5 ug/ml in two separate experiments, conducted using NTP protocols. Actively growing cultures were exposed for 2 or 19-22.5 h, without and with S9, respectively. Cultures exposed without S9 were washed before completing the 19-22.5 h incubation in fresh medium. Colcemid was added to all cultures two hours before the end of the incubation period to arrest the cells in metaphase. Vehicle control and positive controls were similarly treated. Cells were then spread on slides, fixed, stained with Giemsa and 100 cells per culture were scored for chromosome aberrations.

No increases in chromosome aberrations occurred, either with or without S9. The positive controls gave the expected increases in the frequency of aberrations, confirming the sensitivity of the assay.

Ferrocene showed no evidence of inducing chromosome aberrations in Chinese hamster ovary cells when tested at up to 402.5 ug/ml, with and without metabolic activation.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable study performed according to the equivalent of OECD guideline 476; peer-reviewed paper.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium for leukemic cells of mice, supplemented with 10% horse serum, 0.02% pluronic F-68 and 2 mM pyruvate. 0.23% agar was added to the medium for the plates used for the growth phase.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
Without S9: 0, 7.5, 15.0, 18.0 or 21.0 ug Fe/ml
With S9: 0, 1.5, 3.0, 6.0, 9.0 or 12.0 ug Fe/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: listed as an appropriate solvent which at the concentrations used are not cytotoxic to the cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9

Migrated to IUCLID6: 9.1 X 10-5 M
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9

Migrated to IUCLID6: 9.8 X 10-6 M
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (exposure and expression time); in sloppy agar (plate incorporation) (for selection)

DURATION
- Preincubation period: no data
- Exposure duration: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10-12 days


SELECTION AGENT (mutation assays): trifluorothymidine 3 ug/ml

NUMBER OF REPLICATIONS: 2 cultures with 3 plates per culture

NUMBER OF CELLS EVALUATED: 1 X 10(6)/plate for mutants, 200/plate for cloning efficiency

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- Other: colony sizing of the mutants

Evaluation criteria:
Doubling of the mutant frequency over the concurrent solvent treated controls, together with evidence of a dose-related increase, was taken as a positive result.
Statistics:
no data
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data
Remarks on result:
other: strain/cell type: TK+/- (wild-type); TK-/- (mutants)
Remarks:
Migrated from field 'Test system'.

 

Dose (µg Fe/ml)

Rat S9

Absolute cloning efficiency

Relative total growth

(% control)

Average no. TFTR colonies

Mutant frequency/ 106 survivors

Solvent control

0

-

0.67/0.63

100

16/17

26

 

7.5

-

0.55/0.53

63.0

20/25

42

 

15.0

-

0.63/0.55

33.5

41/31

61

 

18.0

-

0.40/0.48

15.5

43/37

93

 

21.0

-

0.29/0.23

5.5

45/49

188

Positive control

 

-

0.41

37.0

72

147

Solvent control

0

+

0.76/0.69

100

31/32

44

 

1.5

+

0.69/0.71

64.5

61/55

83

 

3.0

+

0.74/0.80

78.0

44/51

62

 

6.0

+

0.84/0.72

81.0

40/30

45

 

9.0

+

0.60/0.71

57.5

29/29

45

 

12.0

+

0.59/0.60

34.5

30/38

57

Positive control

 

+

0.48

38.0

101

168

"No increase in the number of small colonies [was observed], and the proportion of large and small colonies remained constant"

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation
negative with metabolic activation

In a reliable FDA and NCI sponsored study, ferrocene exhibited no evidence of mutagenic potential when tested at up to 12 ug Fe/ml, with metabolic activation (S9), in a assay using mouse lymphoma L5178Y cells. However, a dose-related increase in mutant frequency was observed when tested at up to 21 ug Fe/ml, without S9.
Executive summary:

Ferrocene was assessed for its ability to induce gene mutations in an in vitro assay using mouse lymphoma L5178Y cells and conducted to a protocol equivalent to OECD guideline 476.

Cells were treated in duplicate with the test compound, with or without metabolic activation (S9), for 4 h, before washing twice and incubating for 48 h to allow the cells to recover and allow expression of the mutations. Cells were then removed from the suspension cultures and 1 X 106 cells measured into each of three dishes before adding growth medium containing 0.23% agar and incubating for 10-12 days. Solvent and positive control cultures were similarly prepared. Cloning efficiency was accessed by plating 200 cells from each treatment in each of three dishes containing the agar-growth medium without the selective agent.

Ferrocene exhibited no evidence of mutagenic potential when tested at up to 12 ug Fe/ml, with S9, in a assay using mouse lymphoma L5178Y cells (perhaps indicating that S9 metabolises ferrocene to nonmutagenic metabolites). However, a dose-related increase in mutant frequency was observed when tested at up to 21 ug Fe/ml, without S9. No increase was reported in the number of small-sized colonies, indicating that ferrocene induced mainly point mutations, and not chromosomal abberations. The positive controls gave the appropriate increases in mutant frequency, confirming the sensitivity of the assay.

In conclusion, ferrocene induced mutations in mouse lymphoma L5178 cells when incubated in the absence of a mammalian activation fraction.

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 and DPD