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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Man-made mineral fiber hazardous properties assessment using transgenic rodents: example of glass fiber testing.

Author:

Bottin M.C. et al.

Year:

2003

Bibliographic source:

Inhalation Toxicology 15, 1017-1027.

Report date:

2003

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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 Nota Q conditions

EC Number:

926-099-9

Molecular formula:

Not applicable as UVCB

IUPAC Name:

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 Nota Q conditions

Test material form:

solid: fibres

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).

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.