Man-made vitreous (silicate) fibres with random orientation with alkaline and alkali earth oxides (Na2O+K2O+CaO+ MgO+BaO) content greater than 18% by weight and fulfilling one of the CLP Regulation Annex VI Note Q conditions

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The IARC Monograph Working Group on man-made vitreous fibres (MMVFs) reviewed many in vitro genetic toxicity studies of MMVFs and reported that genotoxic effects of MMVFs have been

demonstrated in several cultured cell types, including human cells, and in cell-free assays. However, several caveats can be raised about the in vitro studies on genotoxicity: (i) these assays are short-term

and do not address issues related to fibre dissolution or biopersistence; and (ii) relatively high levels of man-made vitreous fibres on a mass basis have been studied, and the relevance to in vivo exposure

levels is questionable. Many of the reviewed in vitro studies do not specify whether the MMVFs fulfil the Note Q criteria.

It is important to appreciate the degree to which biopersistence plays a role in the different studies and end-points under review, as this property of fibres is thought to be critical in determining chronic toxicity and carcinogenic outcome in humans and in experimental animal systems. In vitro assays are invariably short-term (i.e. from hours to days), and the effect of fibre durability is unlikely to be detected in such assays. Short-term tests could give a misleading impression of possible long-term biological effects. The IARC workin group concluded that this will most likely become manifest as a false-positive result in an in vitro assay for long, highly biosoluble (Note Q) fibres.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

Endpoint:

genetic toxicity in vitro, other

Type of information:

other: IARC monograph

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Principles of method if other than guideline:

This publication evaluates the carcinogenic risks of man-made vitreous fibers in humans with the help of international working groups of experts prepared, critical reviews and evaluations of evidence on the carcinogenicity of a wide range of human exposures.

The IARC Monograph Working Group on man-made vitreous fibres (MMVFs) reviewed many in vitro genetic toxicity studies of MMVFs and reported that genotoxic effects of MMVFs have been demonstrated in several cultured cell types, including human cells, and in cell-free assays. However, several caveats can be raised about thein vitrostudies on genotoxicity: (i) these assays are short-term and do not address issues related to fibre dissolution or biopersistence; and (ii) relatively high levels of man-made vitreous fibres on a mass basis have been studied, and the relevance to in vivo exposure levels is questionable. Many of the reviewed in vitro studies do not specify whether the MMVFs fulfil the Note Q criteria.

It is important to appreciate the degree to which biopersistence plays a role in the different studies and end-points under review, as this property of fibres is thought to be critical in determining chronic toxicity and carcinogenic outcome in humans and in experimental animal systems. In vitro assays are invariably short-term (i.e. from hours to days), and the effect of fibre durability is unlikely to be detected in such assays. [The IARC Monograph Working Group on MMVFs noted that endotoxin is a potent environmental contaminant and its presence in fibre samples could enhance their ability to cause acute inflammation. The presence of endotoxin or the steps taken to inactivate it, were not always reported.] Therefore, short-term tests could give a misleading impression of possible long-term biological effects. This will most likely become manifest as a false-positive result in an in vitro assay for long, highly biosoluble fibres.

Conclusions:

Genetic toxicity in vitro testing might not provide the most appropriate information for assessing the genetic toxicity potential of Note Q MMVFs due to limitations of in vitro studies regarding biopersistence of fibres, perhap resulting in false-positive results of long, highly biosoluble fibres.

Executive summary:

The IARC Monograph Working Group on man-made vitreous fibres (MMVFs) reviewed many in vitro genetic toxicity studies of MMVFs and reported that genotoxic effects of MMVFs have been demonstrated in several cultured cell types, including human cells, and in cell-free assays. However, several caveats can be raised about thein vitro studies on genotoxicity. In vitro assays are invariably short-term (i.e. from hours to days), and the effect of fibre durability is unlikely to be detected in such assays. Therefore, short-term tests could give a misleading impression of possible long-term biological effects. This will most likely become manifest as a false-positive result in an in vitro assay for long, highly biosoluble fibres.

Based on this conclusion from IARC, genetic toxicity in vitro testing might not provide the most appropriate information for assessing the genetic toxicity potential of Note Q MMVFs. In addition, there are available in vivo data on genetic toxicity on Note Q MMVFs.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because adequate data from a reliable in vivo mammalian gene mutation test are available

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Genetic toxicity in vivo

Description of key information

There are 2 in vivo studies considered for the evaluation of genetic toxicity of Note Q man-made vitreous fibres. One study tested CM44 glass fibres, of which the weighted half-life might or might not fulfil the Note Q criteria (due to variability in test results), and the other study tested MMVF 10 glass fibres, which does not fulfil the Note Q criteria but has been shown to have no tumourigenic effects after chronic 2 - year inhalation exposure in rats. Therefore, the data was evaluated in a weight-of-evidence approach.

Both studies used the transgenic BigBlue rats to determine the mutagenic potential of these glass fibres and reported no mutagenic effects. Bottin et al. (2003) reported no increase in mutation frequency up to 90 days following nose-only inhalation exposure to 6.3 mg/m3 CM44 for 5 consecutive days in BigBlue rats. Topinka et al. (2006) reported that MMVF 10, a glass fibre not fulfilling the Note Q criteria but nevertheless demonstrated no tumourigenic potential in rats after 2-year inhalation exposure, triggered DNA strand breaks but no mutagenic effects 16 weeks after intratracheal instillation exposure to 4 weekly doses of 2 mg/animal of this glass fibre.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

fibre biopersistence and mutation in lung DNA

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003-04-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Detection of mutation events in the lung of transgenic rats
- Parameters analysed / observed: Mutant frequencies

GLP compliance:

not specified

Type of assay:

transgenic rodent mutagenicity assay

Specific details on test material used for the study:

The CM44 fiber batch (d = 2.54 g/cm3) used in this study was provided by De Reydellet (Isover, La Defense, France). It was similar in composition to the "C" fiber described in the publication by Bernstein et al. (1996; Inhalation Toxicology 8:345-385).

Species:

rat

Strain:

Fischer 344

Remarks:

Transgenic Lacl F344 (lambda LIZ, BigBlue)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Stratagene (La Jolla, CA)
- Age at study initiation: 3 month old
- Assigned to test groups randomly: Randomly selected animals were sacrified at 1, 3, 14, 28, and 90 days after the beginning of the exposure.
- Housing: housed in polycarbonate cages (1/cage) covered with spun-bonded polyester cage filters
- Diet: pellet food ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1°C
- Humidity (%): 40-60%
- Air changes: air pressure was 5 mm H2O above the atmospheric pressure.
- Photoperiod (hrs dark / hrs light): fluorescent lighting 12 h/day

Route of administration:

inhalation: aerosol

Vehicle:

none

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed within an inhalation chamber previously described by Rihn et al. 1996 (Toxicology 109:147-156).
- Method of holding animals in test chamber: nose-only inhalation; housed in transparent restraining
- Source and rate of air: Clean air was provided by a six-bar compressor that delivered a 100-L/min airstream by an inverted cyclone device. The tangential position of the air inlet pipe gave the airflow a helicoidal movement from the top to the bottom of the cell in order to ensure aerosol homogeneity.
- System of generating particulates/aerosols: The fibers were packed into a cylinder and pushed with a Teflon-coated piston onto a steel brush. The aerosol concentrations were monitored online by photometry.
- Temperature, humidity, pressure in air chamber: Temperature 20-22°C; Humidity 40-60%; Pressure inside the inhalation chamber maintained below the room pressure at 5 mm H2O.
- Air change rate: air volume exchange (100 L) was 60 times/h

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of airborne dust by sampling on PVC filters (GLA-5000, Pall, Saint-Germain-en-Laye, France) for 90 min at a flow rate of 1 L/min, 4 times during each 6-h exposure period. The sampling head was a closed-face Millipore cassette (M000025AO, Millipore, Molsheim, France). The weight of the filters before and after sampling was corrected according to the weight variations of three unexposed filters.
To determine the fiber number, a 15-s sampling period was chosen to avoid filter overload. 0.8-μm mixed ester cellulose membranes (AAW6025C, Millipore) was used to sample the fibers, which were counted by phase-contrast microscopy. The fiber size distribution of either the aerosol or the bulk CM44 sample by scanning electron microscopy (JSM 840-A, Jeol, Tokyo) was assessed. For this measurement, the fibers were deposited on a polycarbonate (110607, Nucleopore, Millipore) filter covered with a thin layer of gold.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

6 h/day

Frequency of treatment:

5 days

Post exposure period:

1, 3, 14, 28 or 90 days after fiber exposure

Dose / conc.:

6.3 mg/m³ air (analytical)

Remarks:

Mean dust gravimetric concentration from 20 filter measurements

No. of animals per sex per dose:

Male animals only
Treatment: 7 per series
Control: 5 per series

Control animals:

yes

Positive control(s):

none

Tissues and cell types examined:

Lung tissue and fluids
- At each time point, bronchalveolar lavage (BAL) fluids by washing the lung 2 times with saline were measured.
- Fiber burden in the lungs and mutagenesis of lung DNA were assessed.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES: Treatment lasted 6 h/day for 5 days. Rats were examined for fiber biopersistence and mutation in lung DNA at 1, 3, 14, 28 and 90 days after fiber exposure.

DETAILS OF SLIDE PREPARATION:
- BAL fluid was spun, May-Grunwald-Giemsa staining was performed, and alveolar macrophages with one or more nuclei were counted.
- Lung DNA was extracted and in vitro packaging was performed (full details in methods section of report).

METHOD OF ANALYSIS: Cytologic and ultrastructural examinations performed for inflammatory response and lung fiber burden; lacI and cll mutagenic assays for mutation assessment

Evaluation criteria:

not specified

Statistics:

Performed for in vivo mutagenesis assay but test not specified

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

The half-time of the fibers >20 μm was 12.8 days. It was mentioned by the authors that glass-wool fibers may not pass the EC Directive noncarcinogenic criterion (i.e. Note Q), i.e. a half-time clearance of less than 10 days from a short-term biopersistence inhalation test. However, the composition of CM44 glass fiber is closely similar to "C" glass fibers tested and described by aforementioned study of Bernstein et al. (1996). For the fibers >20 μm, CM44 has a half-time of 12.8 days compared to the 4.1 days calculated for C fibers in the Bernstein et al. (1996) study. This observation underlines the need to study endpoints (e.g., by biopersistence) in multicentric studies to evidence the source of interlaboratory variation. The half-time shift between CM44 and "C" glass fibers may be explained by at least two factors: (1) Fibers generated in this study were shorter (length = 3.8  ±  2.0  μm) compared to those used previously (length = 13.0  ±  2.3  μm; refer to aforementioned Bernstein et al., 1996 study), and in this study (2) the aerosol contained, on average 1256 particles/cm³, compared to 21 particles/cm³ in the Bernstein et al., 1996 study.

The fibers reached the alveoli as observed in alveolar macrophages in BAL. By 1 and 90 days after exposure, 90% of fibers were shorter than 10.7 and 7.9 μm, respectively. At 90 days, only 20% of total fiber mass remained in the lung (region not known due to digestion protocol).

Mutation assessment via lacI and cll mutagenic assays in BigBlue rats showed no difference in mutations between control and CM44-exposed rats at all time points examined (see table below).

Table: In vivo mutagenesis analysis with either LacI or cII BigBlue systems

		Control		CM44 exposed
Assay	Time [h]	UFPx10^-3	MFx10^5	UFPx10^-3	MFx10^5	IF	p-value
LacI	1	248	1.36	226	2.22	1.64	0.41
	3	278	1.56	250	1.29	0.83	0.77
	28	307	1.72	253	1.45	0.84	0.79
	90	208	2.36	185	2.64	1.12	0.85
cII	1	353	18.14	418	12.09	0.67	0.36
	3	221	15.92	256	15.72	0.99	0.96
	28	315	14.75	321	15.84	1.07	0.85
	90	294	18.54	329	14.71	0.79	0.58

UFP: mean of unit forming plaques numbers analyzed by time point; MF, mutant frequency; IF, induction factor

Conclusions:

Mutation assessment via lacI and cll mutagenic assays in BigBlue rats revealed no difference in mutation frequencies between control and CM44 fiber-exposed BigBlue rats at all time points examined (up to 90 days after 5-day exposure).

Executive summary:

Male transgenic LacI F344 BigBlue rats (3-months-old) were exposed via nose-only inhalation to 6.3 mg/m³ of CM44 glass fibers for 6 h/day for 5 days. Rats were examined for fibre biopersistence and mutation in lung DNA at 1, 3, 14, 28 and 90 days after fiber exposure. The fibers reached the alveoli as observed in alveolar macrophages in BAL. By 1 and 90 days after exposure, 90% of fibres were shorter than 10.7 and 7.9 μm, respectively. At 90 days, only 20% of total fibre mass remained in the lung (region not known due to digestion protocol). Mutant frequencies of control and CM44-exposed rats were similar across all time points.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Remarks:

in vivo mammalian germ cell study (gene mutation) and in vivo mammalian cell study (DNA damage and/or repair)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2005-12-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

- Principle of test: Mutation assay using transgenic (BigBlue) rats; DNA strand breaks via Comet assay
- Short description of test conditions:
1. Homozygous male transgenic λ-lacI F344 rats (Big BlueTM rats) at the age of 10 weeks were used for the assessment. Each cell of these rats carries 30–40 copies of the λ-lacI–α-lacZ shuttle vector (λ-LIZ), which serve as an indicator of gene mutation.
2. BAL macrophages and type II epithelial cells were isolated from the lungs of Fischer 344 rats and used to examine the presence of single-strand breaks using the alkaline comet assay. Comets were analysed by visual scoring of 100 randomly selected images per gel, classifying them into five categories representing increasing relative tail intensity and thus increasing degrees of damage.

GLP compliance:

no

Type of assay:

other: transgenic rodent mutagenicity assay and mammalian comet assay

Specific details on test material used for the study:

Glass wool MMVF10 was obtained from the North American Manufacturer’s Association (NAIMA) repository.

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Homozygous transgenic λ-lacI F344 rats (BigBlue rats) were used to assess mutagenesis and oxidative damage. Each cell of these rats carries 30–40 copies of the λ-lacI–α-lacZ shuttle vector (λ-LIZ), which serve as target for mutagenesis in this mutation analysis system.

Fischer 344 wildtype rats were also used for analysis of DNA single strand breaks, inflammatory markers and histology.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Stratagene (La Jolla, CA, USA)
- Age at study initiation: 11 week
- Diet: standard diet (Altromin) ad libitum
- Water: ad libitum
- Acclimation period: 1 week

Route of administration:

intratracheal

Vehicle:

saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Fibre samples were weighed and suspended in sterile saline to give a concentration of 10 mg/ml. These were sonicated for about 1 min to break any clumps of fibres and immediately prior to injection the suspensions were mixed by rapidly inverting the syringe five times by hand.

Duration of treatment / exposure:

Transgenic (for mutagenesis and oxidative damage assessment): Single doses of 1 or 2 mg/animal, or with four weekly doses of 2mg of fibres per animal (amounting to a total dose of 8 mg/animal)
Wildtype (for single strand breaks, inflammatory markers and histology): A single dose of 2 mg/animal and multiple doses of 4×2 mg/animal (2 mg/week) were employed.

Frequency of treatment:

Single dose or weekly

Post exposure period:

Four or sixteen weeks after the last treatment

Dose / conc.:

1 other: mg/animal

Remarks:

Single dose

Dose / conc.:

2 other: mg/animal

Remarks:

Single dose

Dose / conc.:

8 other: mg/animal

Remarks:

4 weekly doses of 2 mg of fibres

No. of animals per sex per dose:

5 males/dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

lung tissue, regional lymph nodes, BAL macrophages and type II epithelial cells were isolated from the lungs,

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The selection of doses was based on previous experience: the selected doses were not enough to cause observable general toxicity (loss of body weight, etc.) but high enough to give inflammatory reaction in the lung as one of the expected mechanisms of fibre mutagenesis (indirect mechanism).

DETAILS OF SLIDE PREPARATION: The lungs and regional lymph nodes were removed from two animals from each fibre-treated and control groups, fixed in buffered 8% neutral formalin (pH 7.4) and embedded in Paraplast (Sigma, USA). Specimens were histologically evaluated by haematoxylin (H) and eosin (E) staining.

OTHER:
- Malondialdehyde (MDA) levels - MDA in lung tissue was determined by high performance liquid chromatography of the MDA-thiobarbituric acid adduct. Briefly, 0.1 g of tissue was homogenised on ice in 1mL of PBS (without Mg and Ca) and frozen in liquid nitrogen. Samples were kept at −80 °C until analysis. Sodium dodecyl sulfate (0.4 mL of 8% solution), 1 mL of 20% acetic acid and 2 mL of 0.7% thiobarbituric acid was added to 0.4 mL of sample homogenate. MDA – 1,1,3,3-tetraethoxypropane was used as a standard. After 1 h incubation at 100 °C, the samples were cooled in ice and centrifuged at 3500 rpm×15 cm for 10 min. Conditions of HPLC measurement: elution with phosphate buffer pH 6.8: methanol in the ratio 6:4, flow rate 1.5 mL/min, volume of the sample 50 µL, excitation wavelength 532 nm, emission wavelength 553 nm, HPLC column C18 (Waters).
- Biomarkers of lung inflammation: Groups of eight male Fischer 344 were treated with single (2 mg) or multiple doses (4×2 mg) of MMVF 10 fibres. Bronchoalveolar lavage (BAL) was performed and percentage of neutrophils, TNF-α and IL-1α levels were quantified. For determination of TNF-α and IL-1α levels, the cells were resuspended at a concentration of 10E6 AM/mL of BALF. Leucocytes in lavage fluid were cultured in suspension for 20 h in DMEM, and supernatants were frozen at −70 °C until assay. Cytokine concentrations were estimated by appropriate kits [Endogen Rat Tumor Necrosis Factor alpha ELISA and Endogen Rat Interleukin-1 alpha ELISA (ENDOGEN Inc., Woburn, MA, USA)].

Evaluation criteria:

For histology, all lobes were examined and graded according to the Wagner grading scale as suggested by guidelines from the 1982 WHO Conference on Biological Effects of Man-Made Mineral Fibres. According to this scale, Grade 1 is normal, Grades 2–3 involve cellular change, and Grades 4 and above (up to 8) show evidence of fibrosis.

Statistics:

The unpaired Student’s t-test was employed for the statistical analysis of the data. The animal was taken as the statistical unit.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

No significant mutagenesis was caused by MMVF10 fibres despite the generation of significant levels of DNA damage. This suggests that factors in addition to DNA damage are required for mutagenesis detected by Big Blue transgenic assay.

Toxicity:

yes

Remarks:

Significant increase in malondialdehyde (MDA) levels at the highest dose of 4 x 2 mg of MMVF 10 after 16 weeks of exposure; mild toxic and morphologic effects in the lung sections

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Mutagenicity: MMVF10 did not induce a statistically significant increase of mutation frequency at any dose and up to 16 weeks of exposure although the data might suggest a dose-dependent increase of MF.

DNA strand breaks: MMVF 10 induced a significant increase of DNA single strand breaks in alveolar macrophages (AM) and lung epithelial type II cells in BAL at doses 2 mg/animal and 4×2 mg/animal and post-exposure times of 4 and 16 weeks.

Oxidative damage: MDA concentration in lung tissue homogenate was determined as a marker of oxidative damage. The group of animals exposed for 16 weeks to the highest dose of 4×2 mg of MMVF 10 showed significant increase in MDA levels.

Tissue damage: Histological examination of lung sections derived from MMVF 10-treated rats indicates mild toxic and morphologic effects of fibers scored according to the Wagner scale.

Conclusions:

Under the conditions employed in this study, MMVF 10 glass wool fibres induced low levels of inflammation and no detectable mutagenesis.

Executive summary:

Male transgenic (BigBlue) and wildtype Fischer 344 rats (11-weeks-old) were exposed intratracheally to single doses of 1 or 2 mg/animal or to four weekly doses of 2 mg of fibres/animal (amounting to a total dose of 8 mg/animal). Animals were examined 4 or 16 weeks after last exposure. Analysis performed include the evaluation of gene mutations, DNA strand breaks, inflammation and oxidative stress. MMVF10 did not induce a statistically significant increase of mutation frequency at any dose and up to 16 weeks of exposure although the data might suggest a dose-dependent increase of MF. MMVF 10 induced a significant increase of DNA single strand breaks in alveolar macrophages and lung epithelial type II cells in BAL at doses 2 mg/animal and 4×2 mg/animal and post-exposure times of 4 and 16 weeks. The group of animals exposed for 16 weeks to the highest dose of 4×2 mg of MMVF 10 showed significant increase in MDA levels (marker of oxidative damage) in lung tissue homogenate. Histological examination of lung sections derived from MMVF 10-treated rats indicates mild toxic and morphologic effects of fibers scored according to the Wagner scale. It was concluded that under the conditions employed in this study, MMVF 10 glass wool fibres induced low levels of inflammation and no detectable mutagenesis.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Remarks:

in vivo mammalian germ cell study (gene mutation) and in vivo mammalian cell study (DNA damage and/or repair)

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

HYPOTHESIS FOR THE ANALOGUE APPROACH

MMVF 10, a glass wool fibre, was used as read-across (source) substance for the target substances (Note Q man-made vitreous fibres or MMVFs). Mineral wool fibres, including those made of glass, are synthetic fibres that belong to the group of MMVFs and have been shown to be less pathogenic than other MMVFs such as refractory ceramic fibres (RCF) (see IARC Monograph Vol. 81 on synthetic vitreous fibres). One reason for this is that the mineral wool fibres have been demonstrated to be less biopersistent than RCFs. The target substance of MMVFs fulfilling the Note Q criteria under the CLP Regulation (No 1272/2008) are also considered to have lower biopersistence than RCFs.

MMVF 10 has a weighted half-life for inhalation exposure in rats of 37 days, which does not fulfil the Note Q criteria. However, according to the study of Hesterberg et al., 1993 (Fundam Appl Toxicol 20:464-476), tumour incidence was not elevated in male Fischer 344 rats exposed nose-only to three concentrations (3.1, 17.1 and 27.8 mg/m^3) of MMVF10 for 2 years. Given this data and the nearly identical chemical components of MMVF 10 glass wool fibres and Note Q MMVFs, it is considered suitable to consider genetic toxicity data of MMVF 10 for Note Q MMVFs using a read-across approach.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

No significant mutagenesis was caused by MMVF10 fibres despite the generation of significant levels of DNA damage. This suggests that factors in addition to DNA damage are required for mutagenesis detected by Big Blue transgenic assay.

Toxicity:

yes

Remarks:

Significant increase in malondialdehyde (MDA) levels at the highest dose of 4 x 2 mg of MMVF 10 after 16 weeks of exposure; mild toxic and morphologic effects in the lung sections

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Mutagenicity: MMVF10 did not induce a statistically significant increase of mutation frequency at any dose and up to 16 weeks of exposure although the data might suggest a dose-dependent increase of MF.

DNA strand breaks: MMVF 10 induced a significant increase of DNA single strand breaks in alveolar macrophages (AM) and lung epithelial type II cells in BAL at doses 2 mg/animal and 4×2 mg/animal and post-exposure times of 4 and 16 weeks.

Oxidative damage: MDA concentration in lung tissue homogenate was determined as a marker of oxidative damage. The group of animals exposed for 16 weeks to the highest dose of 4×2 mg of MMVF 10 showed significant increase in MDA levels.

Tissue damage: Histological examination of lung sections derived from MMVF 10-treated rats indicates mild toxic and morphologic effects of fibers scored according to the Wagner scale.

Conclusions:

Under the conditions employed in this study, MMVF 10 glass wool fibres induced low levels of inflammation and no detectable mutagenesis.

Executive summary:

Male transgenic (BigBlue) and wildtype Fischer 344 rats (11-weeks-old) were exposed intratracheally to single doses of 1 or 2 mg/animal or to four weekly doses of 2 mg of fibres/animal (amounting to a total dose of 8 mg/animal). Animals were examined 4 or 16 weeks after last exposure. Analysis performed include the evaluation of gene mutations, DNA strand breaks, inflammation and oxidative stress. MMVF10 did not induce a statistically significant increase of mutation frequency at any dose and up to 16 weeks of exposure although the data might suggest a dose-dependent increase of MF. MMVF 10 induced a significant increase of DNA single strand breaks in alveolar macrophages and lung epithelial type II cells in BAL at doses 2 mg/animal and 4×2 mg/animal and post-exposure times of 4 and 16 weeks. The group of animals exposed for 16 weeks to the highest dose of 4×2 mg of MMVF 10 showed significant increase in MDA levels (marker of oxidative damage) in lung tissue homogenate. Histological examination of lung sections derived from MMVF 10-treated rats indicates mild toxic and morphologic effects of fibers scored according to the Wagner scale. The study concluded that under the conditions employed in this study, MMVF 10 glass wool fibres induced low levels of inflammation and no detectable mutagenesis.

This information is used in a read-across approach in the assessment of the target substance (see justification for type of information).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Long-term inhalation carcinogenicity toxicity studies with MMVF note Q fibres are presented in section 7.7. MMVF note Q fibres induced minimal collagen deposition (similar to what could be expected for any biologically inert dust at the same exposure level). No lung fibrosis was observed in the exposed animals. MMVF note Q fibres did not demonstrate a carcinogenic potential neither in the lungs nor in pleura. Furthermore, MMVF note Q fibres are inorganic fibres, whose physicochemical properties suggest a low potential to cross biological membranes, making it unlikely that a mutagenic effect would be found in bacteria or in mammalian cells. There is sufficient evidence to assess MMVF note Q fibres as not mutagenic. Thus, mutagenicity studies are waived because they are scientifically unjustified.

Short description of key information:

It is assessed that MMVF note Q fibres do not possess mutagenic properties, neither in bacteria nor in mammalian cells. MMVF note Q fibres shall not be classified as mutagenic according to the criteria in Council Directive 67/548/EEC and Regulation (EC) 1272/2008.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Mutagenicity studies in bacteria and mammalian cells are waived. The justification is that long-term inhalation studies show that MMVF note Q fibres did not show carcinogenic potential neither in the lungs or pleura. Furthermore, the physicochemical properties of MMVF note Q fibres suggest low potential to cross biological membranes, making it unlikely that a mutagenic effect would be found in bacteria or in mammalian cells. MMVF note Q fibres are assessed to be non-mutagenic, and shall not be classified as mutagenic according to the criteria in Council Directive 67/548/EEC and Regulation (EC) 1272/2008.