Fumes, silica

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read -across to synthetic amorphous silica was applied. In vitro bacterial studies, a mammalian cytogenetic study and a cell transformation study with synthetic amorphous silica have been negative. Comet assays have shown inconclusive results.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read -across to synthetic amorphous silica was applied. An in vivo chromosomal aberration test and a dominant lethal test as well as an ex-vivo hprt mutation study have been negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

10-12 wk old random-bred rats (weight 280-350 g) were fed commercial 4% fet diet and water ad libitum

Route of administration:

oral: gavage

Vehicle:

0,85% saline

Duration of treatment / exposure:

either single administration (acute) or repeated administration (5 times, subacute)

Frequency of treatment:

either single administration (acute) or repeated administration (5 times, subacute)

Post exposure period:

6, 24 and 48 hours after administration (single dose) and 6 hours after last administration (5 doses)

Remarks:

Doses / Concentrations:
1.4, 14.0, 140, 500, 5000
Basis:
nominal conc.
mg/kg

No. of animals per sex per dose:

15 animals per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

yes, 0.3 mg/kg triethylene melamine

Details of tissue and slide preparation:

Bone marrow was retrieved from femur and cell preparations were done.

Evaluation criteria:

The chromosomes of each cell were counted and only diploid cells were analyzed. They were scored for chromatid gaps and breaks,
chromosome gaps and breaks, reunions, cells with greater than ten aberrations, polyploidy, pulverization, and any other chromosomal aberrations which were observed. They were recorded on the currently used forms and expressed as percentages on the summary sheets. Fifty metaphase spreads were scored per animal. Mitotic indices were obtained by counting at least 500 cells and
the ratio of the number of cells in mitosis/the number of cells observed was expressed as the mitotic index.

Statistics:

no data

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information): negative
Silica gel did not induce bone marrow chromosomal aberrations in rat.

Executive summary:

In a valid cytogenetic assay in rats no chromosomal aberrations were seen in bone-marrow cells treated with silica gel once or 5 times at dose levels of 1.4, 14 and 140 mg/kg (Litton Bionetics 1974).

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

10-12 wk old random-bred rats (weight 280-350 g) were fed commercial 4% fet diet and water ad libitum

Route of administration:

oral: gavage

Vehicle:

0.85% saline

Duration of treatment / exposure:

single or repeated 5 times during 5 days

Frequency of treatment:

once or once a day for 5 days

Post exposure period:

Following treatment, the males were sequentially mated to 2
females per week for 8 weeks (7 weeks in the subacute study). Two virgin
female rats were housed with a male for 5 days (Monday through Friday). These
two females were removed and housed in a cage until killed. The male was
rested on Saturday and Sunday and two new females introduced to the cage on Monday. Females were killed using CO2
at 14 days after separating from the male, and at necropsy the uterus was
examined for deciduomata (early deaths), late fetal deaths and total implantations.

Remarks:

Doses / Concentrations:
1.4, 14, 140, 500, 5000
Basis:
nominal conc.
mg/kg

No. of animals per sex per dose:

10 males/dose were treated

Control animals:

yes, concurrent vehicle

Positive control(s):

yes, 0.3 mg/kg triethylene melamine, i.p.)

Tissues and cell types examined:

the uterus was examined for deciduomata, late fetal deaths and total implantations

Statistics:

no data

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Some changes were observed in the low and mid dose group but in the high dose, no significant changes were seen.
Sub acute: In the high dose groups significant decreases were seen in fertility index and number of implants. Dose releated decreases were observed in corpora lutea and dead implants/pregnant female. Dose releated increases were seen in corpora lutea (week 3), preimplantation
loss (week 2, 3). A time trend pattern was not found. However, in the follow up study with 500 and 5000 mg/kg, no changes in fertility index, preimplantation loss and lethal effects on the embryos were seen.

Conclusions:

Interpretation of results (migrated information): negative
Silica gel did not induce dominant lethal mutations.

Executive summary:

A dominant lethal assay conducted in rats did not show significant adverse effects on reproductive performance at the dose levels of 1.4, 14, 140, 500 and 5,000 mg/kg (Litton Bionetics 1974).

Reference 3

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

In a subchronic inhalation study, rats were exposed 6h/d, 5 d/wk to 50 mg SiO2/m3 of hydrophilic pyrogenic silica (Aerosil® 200, MMAD 0.81 µm) or cristobalite (MMAD 1.3 μm) 3 mg/m3 for up to 13 weeks. After the exposure alveolar type II cells were isolated and HPRT mutations frequency was evaluated in vitro. Apoptosis was evaluated by TUNEL staining in histological lung sections.

GLP compliance:

no

Type of assay:

other: HPRT mutations

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 200-250 g

Route of administration:

inhalation: dust

Vehicle:

no vehicle

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Aerosols were generated by a screw-feed mechanism in combination with a venturi -type dust feeder

Controls were exposed to clean air.

Duration of treatment / exposure:

for up to 13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Post exposure period:

HPRT mutation frequences in alveolar type 2 cells and lung cell apoptosis were evaluated after 13 weeks of exposure

Remarks:

Doses / Concentrations:
3 mg/m3 (crystalline silica); 50 mg/m3 (amorphous silica)
Basis:

No. of animals per sex per dose:

4 rats/treatment

Control animals:

yes, sham-exposed

Positive control(s):

quartz

Tissues and cell types examined:

alveolar type II cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: exposure concentration was selected to cause significant inflammatory reaction in lungs
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): see above
DETAILS OF SLIDE PREPARATION: after the termination of exposure, rat alveolar type II cells were isolated by standard methods and freshly isolated alveolar type II cells were seeded into culture flasks and the cells were allowed to attach overnight. The unattached cells were washed away and the cultures were fed every other day with a medium containing 6TG to select for mutation in HPRT gene. After 14-21 days in culture the cells were fixed and immunostained with an antibody to cytokeratins 8, 18, 19 and 6TG resistant cytokeratin staining colonies of >50 cells were counted.
METHOD OF ANALYSIS: Mutation frequaences were counted as (number of colonies/treatment)/(plating efficiency)/10E6 cells =mutants/10E6 cells

Statistics:

Dunnett's test

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Endpoints studied included mutation in the hprt gene of isolated alveolar cells in an ex vivo assay, changes in bronchoalveolar lavage (BAL) fluid markers of cellular and biochemical lung injury and inflammation, expression of mRNA for the chemokine MIP-2, and detection of apoptosis (TUNEL method). After 13 weeks of exposure, the percentage of lavage neutrophils, MIP-2 expression, and lactate dehydrogenase levels as an indicator of cytotoxicity were increased in both silicas. Histopathology of the lungs showed elevated levels of neutrophils and macrophages in lungs and TUNEL staining revealed increased apoptosis. Increased hprt mutation frequency in alveolar epithelial cells was detected with crystalline silica, but not with amorphous silica .

Conclusions:

Interpretation of results (migrated information): negative
Repeated inhalation exposure to synthetic amorphous silica induced lung inflammation but not genotoxicity in rat lungs whereas crystalline silica caused a positive genotoxic response.

Executive summary:

Johnston et al. (2000) conducted a 90-day subchronic inhalation toxicity study with amorphous and crystalline silica. The exposure pattern followed OECD test 413 guidance 'Subchronic inhalation toxicity: 90-day study'. The GLP status was not mentioned. Male Fischer-344 rats (200–250 g) were exposed for 6 h/day, on 5 days/wk, for up to 13 weeks to 3 mg/m³crystalline (cristobalite, mass median aerodynamic diameter 1.3 μm) or 50 mg/m³amorphous silica (hydrophilic pyrogenic Aerosil® 200 Degussa, mass median aerodynamic diameter 0.81 μm). The genotoxic effects on the lung were characterized 13 weeks of exposure. Endpoints included mutation in thehprtgene of isolated alveolar cells in anex vivoassay, changes in bronchoalveolar lavage (BAL) fluid markers of cellular and biochemical lung injury and inflammation, expression of mRNA for the chemokine MIP-2, and detection ofapoptosis. After 13 weeks of exposure, the percentage of lavage neutrophils, MIP-2 expression, and lactate dehydrogenase levels as an indicator of cytotoxicity were increased in both silicas. All parameters remained increased for crystalline silica and decreased rapidly for amorphous silica during the 8-month recovery period. Increasedhprtmutation frequency in alveolar epithelial cells was detected with crystalline silica, but not with amorphous silica. Increased TUNEL staining indicative for apoptosis was seen in macrophages and terminal bronchiolar epithelial cells mainly after exposure to amorphous silica. Lung burdens of silica were 819 and 882 μg for crystalline and amorphous silica, respectively. In summary, genotoxic effects in alveolar epithelial cells occurred only after crystalline but not amorphous silica exposure, despite a high degree of inflammatory response after subchronic exposure to both types of silica. The authors suggest that the additional factors to inflammation, such as biopersistence of particles and direct or direct cytotoxicity to target cells, are important determinants of secondary genotoxic events.

Reference 4

Endpoint:

genetic toxicity in vivo, other

Type of information:

other: IARC review of all available data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

no guideline required

Principles of method if other than guideline:

IARC review of all available data on genetic toxicity of silica

GLP compliance:

no

Type of assay:

other: IARC review of all available data

Species:

other: several

Route of administration:

other: several

Genotoxicity:

ambiguous

Additional information on results:

Crystalline silica dust have induced cell transforming activity in vitro in mammalian cells. High concentrations of crystalline silica have induced DNA damage in cell-free systems and also micronuclei in mammalian cells in vitro. In contrast, after intraperitoneal injection into mice, no micronuclei were detected in vivo. The hprt mutations detected in vitro in the alveolar epithelial cells of rats exposed to crystalline silica dust are probably the result of indirect genotoxic effects. This could be explained by the production of reactive oxygen and nitrogen species which can be formed on reactive SiO2 surfaces or by activation of alveolar macrophages.

Conclusions:

Interpretation of results (migrated information): ambiguous
Even crystalline silica has not been clearly genotoxic and it has been postulated that crystalline silica is more like an indirect genotoxic working through the production of reactive oxygen and nitrogen species which can be formed on reactive SiO2surfaces or by activation of alveolar macrophages.

Executive summary:

In its documentation on silicas IARC (1997) states that "Crystalline silica dust induced cell transforming activity in vitro in mammalian cells. High concentrations of crystalline silica induced DNA damage in cell-free systems and also micronuclei in mammalian cells in vitro. In contrast, after intraperitoneal injection into mice, no micronuclei were detected in vivo. The hprt mutations detected in vitro in the alveolar epithelial cells of rats exposed to crystalline silica dust are probably the result of indirect genotoxic effects. This could be explained by the production of reactive oxygen and nitrogen species which can be formed on reactive SiO₂surfaces or by activation of alveolar macrophages".

Reference 5

Endpoint:

genetic toxicity in vivo, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

No toxicological data were available for silica fume and, therefore, a read-across approach was used. The dissolution, composition and surface properties were the most important parameters considered when deciding which substances can be used for read-across.

Based on the composition, surface characteristics, and bioaccessibility data, silica fume was assumed to have toxicological properties similar to those of sparingly synthetic amorphous silicas. Therefore read-across was carried out using available toxicological studies with synthetic amorphous silica (SAS).

Details on the read-across approach are presented in Iuclid section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vivo and in vitro studies on the mutagenicity of amorphous silicas have not shown relevant positive responses. In one micronucleus study, the positive response quite likely has resulted from the reported high concomitant cytotoxicity. The contradictory results from Comet assays of silicas require more positive backing from results obtained from other methods. The high sensitivity of the assay makes it reproducibility vulnerable to test conditions. Thus, based on weight-of-evidence, amorphous silica is non-genotoxic. According to the comparative in vitro data on the dissolution kinetics in different artificial biological fluids, the dissolution of silicon from silica fume in vitro is similar to synthetic amorphous silica (pyrogenic silica). Thus, there is no reason to assume that silica fume would differ from amorphous silicon dioxide in its mutagenicity.

Crystalline silica, which might occur in trace amounts in bulk material, is not classified as a mutagen. There is data showing that high concentrations of crystalline silica may induce DNA damage in cell-free systems and also micronuclei in mammalian cells in vitro,but these may be a result of indirect genotoxic effects (IARC 1997; SCOEL 2003). Silicon carbide in its non-fibrous form is not a mutagen either.

Other metallic impurities present at levels of >0.1% in silica fume and released at higher amounts from silica fume than from pyrogenic silica mainly include magnesium and zinc, which are not genotoxic elements and do not cause a need to consider the mutagenicity classification of silica fume. Lead levels are usually below 0.1% but low-grade silica fume may contain up to 0.3% of lead (calculated as lead oxide). The classification cut-off limit for germ cell mutagenicity is 0.1% if the compound is classified to CLP categories 1A or 1B for germ cell mutagenicity. According toin vitrorelease data, lead is slowly released from low-grade silica fume particles in vitro. Within the EU, lead is not classified as a mutagen. A recent evaluation by IARC (2006) states:"Lead is a toxic metal and one expression of this property is genetic toxicity. There is, however, little evidence that it interacts directly with DNA at normally encountered blood lead concentrations. The genetic toxicity of lead appears to be mediated in part by increases in, and modulation of, reactive oxygen species. In addition, lead interacts with proteins, including those involved in DNA repair. This latter mechanism might be responsible for enhancing the genotoxicity of other agents. These properties could result in mutation, changes in gene expression and cell proliferation, all of which would contribute to a carcinogenic response if exposure is sustained. "Regarding germ cell mutagenicity, IARC (2003) reports increases in the proportion of morphologically abnormal sperm in mice and cynomolgus monkeys, but not in rabbits, after exposure to soluble lead acetate. Dominant lethal effects were not observed in male mice exposed to lead chloride in a single study. Thus, the evidence on the germ cell mutagenicity of inorganic lead compounds is equivocal. Based on this and the current EU classification of lead compounds, no germ cell mutagenicity classification of silica fume is needed, even in the case of low-grade silica fume containing up to 0.3% of lead. It should be noted that the dissolution of lead from silica fume is, in any case, very low, suggesting it to be in poorly soluble form.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on a read-across to synthetic amorphous silica showing no mutagenicity, no classification for mutagenicity is suggested.