Man-made vitreous (silicate) fibres with random orientation with alkaline oxide and alkali earth oxide (Na2O+K2O+CaO+ MgO+BaO) content greater than 18 % by weight

Carcinogenicity

Administrative data

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Title:

Chronic Inhalation Study of Size-Separated Rock and Slag Wool Insulation Fibers in Fischer 344/N Rats

Author:

McConnell, E. E. et al.

Year:

1994

Bibliographic source:

Inhalation Toxicology,6:6,571 — 614

Materials and methods

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Stone wool fibers MMVF 21

IUPAC Name:

Stone wool fibers MMVF 21

Details on test material:

Rockwool International A/S (Denmark) basalt-based rock (stone) wool (MMVF 21)
Chemical Composition of Bulk Fiber Samples by Weight Percent
SiO2 AI2O3 CaO MgO FeO Fe2O3 Na2O K2O TiO2 MnO S
46.2 13.0 16.9 9.3 4.6 1.8 2.6 1.3 3.0 0.2 0.2

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

The study was conducted at the Research and Consulting Company, Geneva, Switzerland, wing 6-wk old weanling male F344/N rats (Charles River Laboratories, Raleigh, NC). The rats were held for a 2-wk quarantine period, were individually identified using ear tattoos, and were randomly distributed to exposure groups using a computer-generated random algorithm.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

nose only

Vehicle:

clean air

Details on exposure:

The animals were confined separately in tubes that were positioned radially around the exposure chamber (Cannon et al., 1983). This flow-past nose-only system provides a positive pressure laminar flow to each animal individually so that each is supplied fresh aerosol and the air exhaled by one animal does not contaminate the air of others in the chamber (Bernstein & Drew, 1980).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Every 3 mo samples of rock and slag wool aerosols for each dose and crocidolite asbestos were collected on filters for determination of fiber length and diameter. The filter/fiber samples were stored in 100-ml glass bottles containing ~100 ml distilled water and 8 mg sodium azide. Due to their high solubility in water, the last seven slag wool filter samples were stored dry prior to counting. Each filter was removed and rinsed back into the original sample bottle. Suspensions were diluted to 250 ml with distilled water and homogenized by sonification. Aliquots were drawn and filtered onto 0.2 µm Nuclepore and 0.2-µm Millipore membranes. The Nuclepore membranes were dried, applied to scanning electron microscope (SEM) stubs, coated with gold, and counted by scanning electron microscopy (JEOL T-300 or JEOL 840 equipped with a Videoplan image analysis system). The Millipore membranes were placed between glass slides and clarified for optical measurement.
Diameters were measured at 5000x in a minimum of 20 fields or 200 fiber ends as outlined by WHO for counting MMVFs (WHO, 1985). Length distributions were determined at a lower magnification (1500-1850x) according to WHO guidelines (WHO, 1985) using a Leitz Orthoplan or Zeiss optical microscope equipped with a Videoplan image analysis system to allow visualization of entire fibers. A minimum of 20 fields or 200 fiber ends was counted, and the number of truncated ends was recorded. While 40% of the fibers in both the aerosol and recovered from the lungs had diameters less than 0.5 µm, the SEM measurements showed almost no fibers having diameters less than 0.1 µm. Fibers with diameters >0.01 µm can clearly be identified as lines using PCM at 2000x. While diameters obviously could not be accurately measured on fibers that small with PCM, the lengths could accurately be measured for all fibers in the distributions.
Diameter and length distributions were determined using SEM at 5000x and 2000x , respectively. Calculation of all arithmetic and geometric means, medians, standard deviations, and diameter and length classifications from raw data was done using the Videoplan computer system.

Duration of treatment / exposure:

Exposed for 6 h/day, 5 days/wk for up to 24 mo.

Frequency of treatment:

5 days/wk for up to 24 mo.

Post exposure period:

Following the 24-mo exposure period, the animals were held for lifetime observation (until -20% survived), which occurred at 28 mo.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
3.1 mg/m3; 34 WHO fibers/cm3; 13 fibers L>20 µm/cm3
Basis:
analytical conc.
SEM Bivariate analysis

Doses / concentrations 2

Remarks:

Doses / Concentrations:
16.1 mg/m3; 150 WHO fibers/cm3; 74 fibers L>20 µm/cm3
Basis:
analytical conc.
SEM Bivariate analysis

Doses / concentrations 3

Remarks:

Doses / Concentrations:
30.4 mg/m3; 243 WHO fibers/cm3; 114 fibers L>20 µm/cm3
Basis:
analytical conc.
SEM Bivariate analysis

No. of animals per sex per dose:

140

Control animals:

yes, sham-exposed

Details on study design:

This study was designed to investigate the potential pathogenic effects in Fischer 344/N rats of two different types of man-made vitreous fibers (MMVF). Eight-week-old male rats were exposed in nose-only inhalation chambers, 6 h/day, 5 days/wk, for 24 mo to 3 concentrations (3, 16, and 30 mg/m’) each of the two MMVFs: a basalt-based rock wool (stone wool), and a slag wool (blast furnace). Crocidolite asbestos (10 mg/m’) was used as a positive control. The experimental groups were compared to unexposed (chamber) controls. The MMVFs used in this study were size selected to be largely respirable in rats. Interim sacrifices took place at 3- and 6-mo intervals to monitor the progression of pulmonary changes. Fibers were recovered from digested lung tissue for determination of changes in fiber number and morphology.

Positive control:

Crocidolite asbestos

Examinations

Observations and examinations performed and frequency:

Clinicopathology:
The rats were observed daily for clinical signs, morbidity, and mortality throughout the study. They were individually examined and weighed once
each week during the first 13 wk and at least once each month thereafter.

Aerosol Monitoring and Characterization
Fiber mass concentrations were measured at least 4 times/wk during the 2-yr exposure period.
Every 3 mo samples of rock and slag wool aerosols for each dose and crocidolite asbestos were collected on filters for determination of fiber length and diameter by scanning electron microscopy.

Sacrifice and pathology:

A necropsy was performed on all animals, and a complete set of tissues (NTP, 1984 ) was obtained and fixed in 10% neutral buffered formalin, except for the lung. The lungs were removed, weighed, and examined under a dissecting microscope. The right accessory lobe was ligated, removed, weighed, and frozen at -20°C for lung fiber retention analysis. The remaining lung was perfused with Karnovski’s fixative via the trachea and fixed for routine histopathology examination. The lungs were examined and classified histopathologically and given a Wagner score for inflammatory change and fibrosis (McConnell et al., 1984).

Other examinations:

At necropsy the accessory lobe of the lung from each animal was removed, weighed, and frozen. After thawing it was dehydrated in acetone and evaporated to constant weight. The dry lung tissue was then ashed in an LFE LTA 504 multiple chamber plasma unit. The ash from each lung was dispersed in distilled water, and aliquots of the suspension were filtered onto 25-mm diameter, 0.2-µm pore size Nuclepore membranes for SEM stub preparation and onto 25-mm diameter, 0.2-µm pore size Millipore membranes that were clarified for optical slides. Counting and measuring of the fibers were done in the same way as with the aerosol fiber samples.

Statistics:

Pairwise comparisons of tumor incidence between exposure groups were made using Fisher’s exact test, and tests for trend were made using an exact algorithm for the Cochran-Armitage test. Only rats exposed to rock or slag wool for 12 mo or crocidolite for 10 mo were considered at risk for the induction of neoplasms, because this was the earliest time point a neoplastic finding was observed in this series of studies. All tests of significance were two-tailed, with no formal adjustment for multiple comparisons. Student’s t-test was used for comparisons of the physical characteristics of the fibers.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal clinical signs were observed in any of the rock or slag wool exposure groups during the course of the study.

Mortality:

no mortality observed

Description (incidence):

No abnormal clinical signs were observed in any of the rock or slag wool exposure groups during the course of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain and survival were comparable to the unexposed controls.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No differences in lung weights (compared to controls) were found at any time point in the 3 mg rock groups or in any of the slag-wool-exposed rats. No Iiing weight differences were observed at 24 mo in any of the groups

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic evidence of few similarly appearing small (1 -2 mm diameter) greyish-white foci on the lung. No macroscopic evidence of pleural changes was noted at any point.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

dose-reIated increase in puI monary macrophages and developing microgranulomas in the alveolar duct region. Mild bronchiolization was noted in all high-dose rats. At 18 mo minimal & mild fibrosis was noted in all rats in the mid- and high dose.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Microscopically , a dose-related increase in pulmonary macrophages and developing microgranulomas in the alveolar duct region were observed at 3 mo. Additionally, a mild amount of bronchiolization was noted in all high-dose rats. The pulmonary response had progressed at 6 mo with bronchiolization being found in the mid- as well as the high-dose group rats. The lesions further progressed at 12 mo, with 2 of 6 rats in the high dose (30 mg) showing a minimal amount of fibrosis in the proximal portion of the alveolar duct region. At 18 mo minimal (16 mg) and mild (30 mg) fibrosis was noted in all rats in the mid- and high-dose groups, although it was more apparent in the latter animals. No significant increase in severity was found at the end of the exposure period (24 mo) nor at the end of the study (28 mo). The number of pulmonary macrophages decreased dramatically during the non-exposure period. Fibers were noted in macrophages, in microgranulomas, and on surfaces of the alveolar ducts and proximal alveoli throughout the study. Additional fibers and especially fiber fragments were observed in macrophages in the peribronchial lymphoid tissues. No treatment-related lesions were observed in the pleura.

Relevance of carcinogenic effects / potential:

There was no evidence of carcinogenic activity following inhalation of high levels of Stone Wool.

Any other information on results incl. tables

Pulmonary Cellular Change and Fibrosis in Male F344/N rats Exposed to Rock or Slag Wool Fibers for 3, 6, 9, 12, 15, 18, or 24 mo

Exposed/sacrificed	Control	Crocidolite		Rock wool			Slag wool
			3 mg	16 mg	30 mg	3 mg	16 mg	30 mg
3/3	1.0	4.0	2.0	2.2	3.2	2.0	2.5	2.5
6/6	1.0	4.2	2.0	2.7	3.3	2.0	2.7	3.0
10/10		4.0b	-	-	-	-	-	-
12/12	1.0	4.0b	2.0	2.7	3.3	2.5	2.2	3.0
18/18	1.0	4.0b	2.5	4.0	4.0	2.7	2.5	2.7
24/24	1.0	4.0b	2.2	4.0	4.0	2.0	2.2	2.8
Recovery
3/24	1.0	4.0	1.3	1.0	1.0	1.3	1.3	1.3
6/24	1.0	4.0	1.0	3.0	2.0	2.0	1.3	2.0
12/24	1.0	4.0 b	1.3	3.3	4.0	1.5	2.0	2.5
18/24	1.0	-	2.0	4.0	4.0	2.2	2.3	2.2
24/28	1.0	4.0 b	2.2	4.0	4.0	2.0	2.6	2.5

 

.Note. -, No animals sacrificed

"(a Average Wagner scores (n = 3 -6) (McConnell etal, 1984.

.b Crocidolite rats were exposed for only 10 mo.

Applicant's summary and conclusion

Conclusions:

In conclusion, these studies demonstrate that the inhalation of high levels of rock (stone) and slag wools, while causing nonspecific inflammatory lesions (rock and slag wool) and fibrosis with high lung burden of long fibers (rock wool only) in the lungs of exposed rats, showed no evidence of carcinogenic activity. These results suggest that respirable fractions of these MMVFs should not pose a significant health risk to humans at the relatively low airborne levels found in the workplace.

Executive summary:

These studies demonstrate that the inhalation of high levels of rock (stone) and slag wools, while causing nonspecific inflammatory lesions (rock and slag wool) and fibrosis with high lung burden of long fibers (rock wool only) in the lungs of exposed rats, showed no evidence of carcinogenic activity. These results suggest that respirable fractions of these MMVFs should not pose a significant health risk to humans at the relatively low airborne levels found in the workplace.