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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
The toxic effects in mice and rats of a 90 day inhalation exposure to ferrocene
Author:
Nikula KJ et al
Year:
1993
Bibliographic source:
Fundam Appl Toxicol 21 (2): 127-139 (1993)

Materials and methods

Principles of method if other than guideline:
Guidelines data not available. A study was made of the toxic effects in mice and rats of a 13 wk inhalation exposure to ferrocene according to methods published by Harkema et al., 1982
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Ferrocene
EC Number:
203-039-3
EC Name:
Ferrocene
Cas Number:
102-54-5
Molecular formula:
C10H10Fe
IUPAC Name:
iron(2+) dicyclopenta-2,4-dienide
Test material form:
other: vapour
Details on test material:
material obtained from Midwest research institute

Test animals

Species:
other: rat and mice
Strain:
other: F344/N rats and B6C3F1 mice
Sex:
male/female
Details on test animals or test system and environmental conditions:
animals from Simonsen labs Inc.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
F344/N rats and B6C3F1 mice were exposed to 0, 3.0, 10, and 30 mg ferrocene vapor/m3
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hr/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
3.0 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
10 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
30 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:
Observations of rats were made after 5 days, 23 days and 13 weeks of exposure. Mice were not evaluated
Other examinations:
The respiratory function of male rats from each of the three exposed groups and one control group was measured after 30, 60, and 90 days of exposure according to previously published methods (Harkema et al.. 1982). These measurements included multiple assays of breathing patterns, lung volumes, mechanical properties oflung tissue, gas distribution uniformity, air flow limitation, and alveolar-capillary gas exchange efficiency. The tests were nondestructive, requiring only halothane anesthesia, and thus were performed serially on the same rats at each time.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Details on results:
Although histopathological lesions were observed in the nose, larynx, trachea, lung and liver of rats and mice and in kidneys of mice, the most severely affected tissue in both species was the nose. The severity of lesions was probably related to the high retention of ferrocene-introduced iron in the nose (Dahl and Briner, 1980) resulting from the high metabolic capacity of this tissue for ferrocene as a substrate (Sun et ai., 1991). Ferrocene is metabolized to hydroxyferrocene (Hanzlik et ai., 1978) which would decompose readily to release ferrous ion intracellularly. The ferrous ion could catalyze lipid peroxidation (Dumelin and Tappel, 1977) or hydroxyl-free radical formation by the Fenton reaction (Hewitt et aI., 1991). Either products of lipid peroxidation or the resulting free radicals could react with key cell components to account for the lesions observed.

The fact that the nasal olfactory mucosa was more affected than the nasal respiratory mucosa is probably due to the higher metabolic activity in the olfactory mucosa, which has been demonstrated in rats for ferrocene (Sun et aI., 1991). Other factors that might have contributed to the greater sensitivity of the olfactory compared to the nasal respiratory mucosa may include a higher tissue dose and a lower rate of chemical clearance. The nasal respiratory epithelium of mice was more affected by ferrocene exposure than that of rats. The metabolic capacity for ferrocene of the olfactory versus respiratory mucosa has not been examined in mice, nor has an interspecies comparison been made. Nasal olfactory versus nasal respiratory deposition of ferrocene has not been determined in either species. However, it has been shown that the mouse has a greater surface area of respiratory epithelium available for filtering air per unit volume of the nasal cavity than the rat (Gross el ai., 1982). This relative increase in area at risk might account for the observed differences in nasal respiratory lesions Nasal epithelial lesions have been classified according to whether they are induced by direct- or indirect-acting chemicals (Gaskell, 1990). Indirect-acting chemicals are metabolized to a toxic intermediate by the mixed-function oxidases known to be present in the olfactory mucosa (Hadley and Dahl, 1983; Dahl and Hadley, 1991). Direct-acting chemicals are those where the parent compound is toxic. The damage caused by ferrocene is similar to that caused by other indirect-acting chemicals, such as 3-trifluoromethylpyridine and 3-methylindole, in that all or a large percentage of the olfactory mucosa was affected, while the respiratory epithelium was relatively spared. Direct-acting chemicals, in contrast, usually cause injury with an anterior-posterior gradient of damage within the nose. The respiratory epithelium is damaged, while olfactory lesions, if they occur, are often restricted to the dorsal meatus (Gaskell, 1990).

The histopathologic findings in the 13-week study differ from those in our earlier 2-week study (Sun el a/., 1991) in that tissues and organs other than the nose accumulated inhaled ferrocene, as suggested by the histochemical stains for iron. Histopathologic lesions in these other tissues and organs were limited to pigment accumulation with degeneration of individual cells. This may reflect a lower capacity for ferrocene metabolism relative to olfactory tissue (known to be the case for rat liver (Sun et al, 1991), low penetration of the ferrocene vapor into those regions (Dahl and Briner, 1980), or the inability to detect subtle membrane damage using light microscopy. Furthermore, the cells in these other sites may have adequate intracellular mechanisms to protect against lipid peroxidation induced by low levels of intracellular iron. Higher levels of intracellular iron may be required to overwhelm these protective mechanisms and to produce lesions observable by light microscopy. The fact that pigment accumulation with concomitant degeneration was observed in individual cells of the larynx, trachea, and bronchioles of both species, and the tit liver of mice, suggests that these cells can hydroxylate ferrocene to some degree, and that cell injury will occur in the cells that accumulate higher levels of ferrocene-derived iron.
The amount of ferrocene swallowed during or after exposure, including that ingested after mucociliary clearance and after fur licking, was not measured..

In summary, several toxicological responses were observed in F344/N rats and B6C3F1 mice after a 13-week inhalation exposure. to ferrocene vapor at nominal concentrations of 3.0, 10, and 30 mg/m3. These included exposure- related but minimal changes in body weights and organ weights and histopathological lesions in the larynx, trachea, lungs, liver, (kidneys only in mice), and most notably in the nasal epithelium. The effects of ferrocene exposures on organ weights may indicate a secondary response to the loss of appetite from the severe nasal lesions or may indicate that these are target organs for chronic toxicity (particularly the liver). In the studies reported here, as in our earlier 2-week study (Sun et al., 1991), the nasal lesions were present in both sexes of rats and mice at all exposure concentrations. It is important to note that the two lowest concentrations that resulted in these lesions were at and below the current TL V for ferrocene (10 mg/m3). Only a chronic study could determine the potential carcinogenicity of inhaled ferrocene vapor.

Effect levels

Key result
Dose descriptor:
LOAEC
Effect level:
3 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: Liver weight

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a peer reviewed study report, no clinical signs were seen in any rats exposed to any dose. The mean iron lung burden in rats exposed to 30 mg/m3 for 90 days was 4 times greater than the burden in control rats. The relative liver weight of rats showed a dose-related increase, which was significant (p<0.05) at the highest dose level for the males and at the 2 highest dose levels for the females. The relative liver weight of female mice was decreased at 3.0 mg/m3, but there was no dose-response relationship. On the other hand, the absolute liver weight of female mice showed a dose-related decrease, which was significant at all dose levels (p<0.05). The effects of ferrocene exposures on organ weights may indicate a secondary response to the loss of appetite from the severe nasal lesions or may indicate that these are target organs for chronic toxicity (particularly the liver).
No exposure-related changes in respiratory function, lung biochemistry, bronchoalveolar lavage cytology, total lung collagen clinical chemistry, and haematological parameters were observed. There w ere neither indications of developing pulmonary fibrosis nor of any haematological toxicity. Exposure-related histological alterations, primarily pigment accumulations, were observed in the nose, larynx, trachea, lung, and liver of both species, and in the kidneys of mice. Lesions were most severe in the nasal olfactory epithelium, where pigment accumulation, necrotising inflammation, metaplasia, and epithelial regeneration occurred. Nasal lesions were observed in all ferrocene-exposed animals and differed only in severity which was dependent on the exposure concentration. The results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by either iron-catalysed lipid peroxidation of cellular membranes or the iron-catalysed Fenton reaction to form hydroxyl radicals that directly react with other key cellular components, such as protein or DNA
The material can be considered to have an LOAEL of 3 mg/m3 air