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

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The two fibre types tested complies with the definiton of FMMVF fibres. The experimental setup, chronic inhalation animal model is considered the most relevant laboratory model for evaluating the potential risk of airborne fibres to human health.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1994

Materials and methods

Principles of method if other than guideline:

The study was performed as nose-only inhalation study with three dose levels (3, 16 and 30 mg/m3) for both MMVF 21 and MMVF 22, only 10 mg/m3 for positive control crocidolite asbestos. The study was performed according to the method described in Sachsse, K.; Ullman,L.; and Zbinden, K.; 1976; Toxikologische Prüfungen von aerosolen in Tierexperiment. Chem. Rundsch. 29(38):Seite1.

GLP compliance:

not specified

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

MMVF 21

IUPAC Name:

MMVF 21

Constituent 2

Reference substance name:

MMVF 22

IUPAC Name:

MMVF 22

Constituent 3

Reference substance name:

Rock wool

IUPAC Name:

Rock wool

Constituent 4

Reference substance name:

Slag wool

IUPAC Name:

Slag wool

Constituent 5

Reference substance name:

FMMVF

IUPAC Name:

FMMVF

Constituent 6

Reference substance name:

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

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report): MMVF 21 and MMVF 22

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at study initiation: six weeks
- Weight at study initiation: No information
- Fasting period before study: No information
- Housing: Hazelton 2000 polycarbonate cages, with hardwood bedding
- Diet (e.g. ad libitum): Pelleted feed, Kliba 343, Klingentalmuehle, AG, Switzerland ad libitum, only during non-exposures period
- Water (e.g. ad libitum):Filtered freshwater ad libitum, only during non-exposures period
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C):No information
- Humidity (%): No information
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light):No information

IN-LIFE DATES: From: six weeks To: 26 months (study period was 24 months, acclimation, 2 weeks)

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Se table in the box "Any other information on materials and methods, including tables"

Details on inhalation exposure:

The animals were confined separately in tubes that were positioned radially around the exposure chamber. This flow-past nose-only system provides a positive pressure laminar flow to reach animals 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Fiber mass concentration were measured at least 4 times per week during the 2 year exposure period. Each aerosol sample from each fiber type and dose level, as well as the control aerosols, was collected on a Gellman membrane filter from one of the laminar flow ports in the exposure chambers to eliminate isokinetic sampling bias. Additionally, aerosol concentrations were monitored continuously during the exposure period using a RAS (GCA Corp.) light-scattering monitor to assure uniformity. The aerosols were analyzed for WHO fibres per cubic centimeter (Defined as having a diameter < 3 µm, a length > 5 µm, and an aspect ratio > 3:1 (WHO,1985)) once each exposure week during week 1-13, and every other week thereafter through exposure week 104. In addition , the total number of fibres and non-fibrous particles were counted once each month on a filter for each fiber type and dose. Samples were collected on Millipore filters, placed between glass slides, and counted by phase-contrast microscopy at 400 X magnification.
Each month total fibres (aspect ratio >3:1) and particles were counted to determine total fibres per cubic centimeter and particles per cubic centimeter.

Duration of treatment / exposure:

six hours a day, 5 days a week for up to 24 months
Groups of 3 to 6 rats were removed from each group from exposure at 3, 6, 12 and 18 month and held until 24 month to determine progression/regression of lesions, and to determine lung fiber burdens following cessation of exposure. Following the 24 month exposure period the animals were held for lifetime observation, untill approximately 20 % survived, which occured at 28 month.

Frequency of treatment:

six hours a day, 5 days a week for up to 24 months

No. of animals per sex per dose:

140 all males

Control animals:

yes, concurrent no treatment

Details on study design:

See under section 7.7 Carcinogenicity; Endpoint study record: Carcinogenicity McConnell et al 1994 below

Positive control:

Crocidolite

Examinations

Observations and examinations performed and frequency:

Lung burden analysis was done in following time points (week of sacrifice/exposure);
For positive control crocidolite; 13/13, 26/26, 44/44, 53/44, 78/44, 104/44, 124/44, 104/26, and 104/13, giving the following recovery periods in weeks; 0, 0, 0, 9, 34, 60, 80, 78, and 90, respectively.
For air control (week of sacrifice/exposure); 13/13, 26/26, 44/44, 53/44, 78/44, 104/44, and 124/44, giving the following recovery periods in weeks; 0, 0, 0, 9, 34, 60 and 80, respectively.
For the two tested fibres (week of sacrifice/exposure); 13/13, 26/26, 53/53, 78/78, 104/104, 124/104, 104/78, 104/53, 104/26 and 104/13, respectively, giving the following recovery periods in weeks; 0 ,0, 0, 0, 0, 20, 20, 51, 78 and 91, respectively.

Clinical observations, body weight and survival.

Lung weight

Sacrifice and pathology:

Animals 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 weeks and at least once every month thereafter. Sick or moribund animals were killed following deep anesthesia by intraperitoneal injection of pentobarbital sodium, as were those remaining at the end of the study.
A necropsy was performed on all animals, and a complete set of tissues was obtained and fixed in formalin, except for the lung that were removed, weighed, and examined under dissection microscope. The right accessory lobe was ligated, removed and frozen for fibre retention analysis. The remaining lung was perfused with Karnovski´s fixative via the trachea at 30 cm H2O pressure for 2 hours. After fixation, a consistently uniform transverse section (2mm diametr) of the left lung and right diaphragmic lobe was obtained from each animal for routine histopathology. In addition, sections were made from all grossly visible lesions in the lung. Replicate sections were routinely stained with hematoxylin and eosin and Masson-Goldner`s trichome stain to highlight collagen.
Other tissues processed routinely for histopathological examination included three levels of the nasal cavity (anterior, midlevel and posterior), larynx, trachea, bronchi, mediastinal and mesenteric lymph nodes, liver, spleen, kidneys, heart and all grossly visible lesions.
The lungs were examined and classified histopathologically and given a Wagner score for inflammatory change and fibrosis

Other examinations:

Lung burden analysis.
The average lung burdens of WHO fibers reached their peak at 1 year and plateaued during the reminder of the study. WHO fiber levels were higher for rock wool than slag wool at various time points during the study, for example, at week 53. 30mg/m3 rock wool= 319,000 ± 53,000 (fibers> 20µm length = 55,000 ±19,000) versus 30 mg/m3 slag wool= 225,000 ±59,000 (fibers> 20µm length = 5,000 ±3000)
The retention of both rock and slag wool fibers decreased rapidly after exposure ceased.
For 18 months exposure and six month recovery, the average number of WHO fibers per milligram dry lung decreased from 230,000 and 283,000 to 47,000 and 116,000 for slag and rock wool, respectively.

No abnormal clinical signs were observed in any of the rock or slag wool exposed groups. Body weight gain and survival were comparable to unexposed controls, whereas the positive control failed to gain weight.

Lung weight.
For the positive control a significant increase in lung weight was observed from 3 month a throughout the experiment. At 18 months the lungs of positive control were 43% heavier than unexposed control.
The lungs of the 30 mg and 16 mg rock wool exposed rat were 24 % and 13 % heavier than unexposed control at 18 month, respectively. No significant difference were observed for the 3 mg rock woll or for the animals exposed to any of the slagwool concentrations.

Statistics:

Pairwise comparisons of tumor incidence between exposure groups were made using Fisher`s exact test.
Test for trend were made using an exact algorithm for the Cochran-Armitage test.
All tests for significance were two-tailed, with no formal adjustment for multiple comparisons.
Student`s t-test was used for comparison of the physical characteristics of the fibres

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increase in lung weigth at 16 mg/m3. For results on lung weights see field other examinations above

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A dose-related increase in microgranulomas in the alveolar duct region were observed at 3 month. A mild bronhiolozation was noted in all high-dose rats. The pulmonary response had progressed at sixth month with bronchiolization found in mid and high

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

For stone wool no statistical significant increase nor a dose-response relationship. No mesothelioma was observed, for slag wool Occasional mesotheliomas were observed but no significantly different from unexposed contro

Details on results:

For Rock wool; A dose-related increase in microgranulomas in the alveolar duct region were observed at 3 month. A mild bronchiolization was noted in all high-dose rats. The pulmonary response had progressed at sixth month with bronchiolization found in mid and high dose. The lesions further progressed at 12. month, with 2 of 6 rats in the high dose showing a minimal amount of fibrosis in the proximal part of the alveolar duct region. At 18. month minimal and mild fibrosis was noted in all rats in the mid and high dose groups, although it was more apparent in the high dose. No significant increase in severity was found at the end of the exposure period, nor at the end of the study. The number of pulmonary macrophages decreased rapidly during non-exposure period. Occasional bronchoalveolar neoplasms were found in all dose groups, including the unexposed control, but there was neither statistically significance, nor a dose-respons relationship.

For Slag wool; There was a dose and time related increase in pulmonary macrophages, microgranuloma formation, and bronchiolization. However, there was no evidence of fibrosis at any point. Fibers and fragments were readily found in many of the macrophages and microgranulomas. No treatment related lesions were observed in the pleura. Occassional neoplasms were found; 2 of 116 in low dose, 0 of 115 in mid dose, and 3 of 115 in the high dose, compared to 2 of 126 in unexposed control

No abnormal clinical signs were observed in any of the rock or slag wool exposed groups. Body weight gain and survival were comparable to unexposed controls, whereas the positive control failed to gain weight.

Lung weight.
For the positive control a significant increase in lung weight was observed from 3 month a throughout the experiment. At 18 months the lungs of positive control were 43% heavier than unexposed control.
The lungs of the 30 mg and 16 mg rock wool exposed rat were 24 % and 13 % heavier than unexposed control at 18 month, respectively. No significant difference were observed for the 3 mg rock woll or for the animals exposed to any of the slagwool concentrations.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

Carcinogenicity

Effect level:

> 30 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Corresponds to 243 fibres per cm3

2

Dose descriptor:

NOAEL

Remarks:

Fibrosis

Effect level:

ca. 3.1 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Corresponds to 34 fibres per cm3

3

Dose descriptor:

LOAEC

Effect level:

ca. 16 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Corresponds to 150 fibres per cm3

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

See under section 7.7 Carcinogenicity; Endpoint study record: Carcinogenicity McConnell et al 1994 below

Applicant's summary and conclusion

Conclusions:

The study was performed as a nose-only inhalation study on Male F 344/M rats. The study is highly relevant for the evaluation of human health, as the exposure route is inhalation, which is considered the most relevant route of concern for human health. The animal used, rats has a lung that are very much like the human lung. The doses selected were approximately 100 to 1000 times higher than what can be expected in the working environment, depending on the actual situation.
For rock wool, a dose-related increase in microgranulomas in the alveolar duct were observed at 3 month, the pulmonary response progressed further at 12. month. At 18. month minimal and mild fibrosis was observed in mid and high dose groups. No significant increase in severity was found at the end of the exposure, nor at the end of the study.The number of pulmonary macrophages decreased rapidly during non-exposure period. Occasional bronchoalveolar neoplasms were found in all dose groups, including unexposed control. No statistical significance was demonstrated, nor a dose relationship.

Slagwool; There was a dose and time related increase in pulmonary macrophages, microgranuloma formation, and bronchiolization. However, there was no evidence of fibrosis at any point. Fibers and fragments were readily found in many of the macrophages and microgranulomas. No treatment related lesions were observed in the pleura. Occassional neoplasms were found; 2 of 116 in low dose, 0 of 115 in mid dose, and 3 of 115 in the high dose, compared to 2 of 126 in unexposed control.

Finally it can be concluded from the study that in very high doses the MMVF 21 and MMVF 22 fibers give rise to increase in pulmonary macrophages as well as any other particulate, but in a reversible manner, when exposure is ceased. Occasional bronchoalveolar neoplasms were found in all dose groups, including the unexposed control. No mesotheliomas were observed in the fiber exposed groups. There was no statistical evidence for a carcinogenic effects of the two fiber types studied.

Executive summary:

see section 7.7 Carcinogenicity; Endpoint study record: Carcinogenicity McConnell et al 1994