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

Description of key information

MMVF note Q fibres are assessed to possess no toxic properties after repeated oral, dermal or inhalation exposure. MMVF note Q fibres shall not be classified according to the criteria in Council Directive 67/548/EEC and Regulation (EC) 1272/2008.

There are a number of repeated dose toxicity rodent studies available on Note Q man-made vitreous fibres (MMVFs), in which fibre lung burden and toxicity effects in the lung have been evaluated. MMVFs in general are more biosoluble than biopersistent fibres such as asbestos (e.g. crocidolite and amosite). In addition, studies have demonstrated the lower lung burden of Note Q MMVFs (e.g. with weighted half-lives of fibres with length > 20 μm after inhalation of less than 10 days) compared to other MMVFs not fulfilling the Note Q criteria under CLP Regulation (i.e. longer weighted half-lives than Note

Q MMVFs).

MMVF 10, a glass wool fibre, was used as read-across (source) substance for Note Q MMVFs. Even though MMVF 10 has a weighted half-life (for fibres >20 μm) for inhalation exposure in rats of 37 days, which does not fulfil the Note Q criteria, a carcinogenicity study showed that tumour incidence was not elevated in male Fischer 344 rats exposed nose-only to three concentrations (3.1, 17.1 and 27.8 mg/m³) of MMVF10 for 2 years compared to control. 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 repeated dose toxicity data of MMVF 10 for Note Q MMVFs using a read-across approach. MMVF10 was demonstrated to have a maximum tolerated dose (MTD) of 30 mg/m3 in rats. Exposure to MMVFs above this MTD resulted in lung overload effects in the rats, meaning that these overload effects are not attributable to the chemical composition of Note Q MMVFs.

The main effects reported among these repeated dose studies (tested up to the MTD of 30 mg/m3) include slight to minimal morphological changes in the lung and slight increase in inflammatory responses in alveolar macrophages of Note Q MMVF-exposed animals. Majority of these effects were shown to be transient as they were no longer observed after cessation of fibre exposure. No fibrogenic potential of Note Q MMVFs was observed in this studies.

In a short-term inhalation study, male Fischer 344 rats were exposed to four glass wools (JM 901/MMVF10.1, JM 901F, JM 902, JM 475; deemed as Note Q MMVFs) and amosite asbestos (positive control) for 5 days, 6 hours/day. The measured concentration of the fibres ranged between 12 to 32 mg/m³. The main effect reported after exposure was increased inflammatory responses in the form of fibrecontaining microgranulomas in JM 901-esposed rats and fibre-containing alveolar macrophages in JM 901 or 902-exposed rats seen 1 day after exposure ended. The effects were no longer observed after 10 and 30 days of recovery without fibre exposure.

In a subchronic inhalation study, male Wistar rats (32 per dose) were exposed nose-only to new biosoluble high-aluminum low-silica HT type stone wool (RIF41001 and RIF42020-6; deemed as Note Q MMVFs) in air for 6 hours/day, 5 days/week for 3 months, and followed post-exposure for several months. After exposure of rats to these biosoluble HT fibers, no biologically significant effects were observed except that a statistically significant increase in lung weight was observed up to 1.5 months post-exposure in all 3 treatment groups. At 3 months post-exposure, the small increase was no longer significant. Minimal morphological changes were diagnosed in the HT fibre groups at 3 months and 1.5 and 3 months post-exposure. No fibrogenic potential noted of the two HT fibres. No clear-cut difference between the different biosoluble fibre types noted. Only slight macrophage reaction seen in lungs of rats exposed to HT fiber types: Wagner grade 1 or 2 in all animals examined.

In a chronic inhalation study, male Fischer 344/N rats were exposed nose-only to slag wool MMVF22 (deemed as Note Q MMVF) in air for 6 hours/day, 5 days/week for 24 months. No abnormal clinical signs, changes in body weight or mortality were observed. Non-specific inflammatory response, no evidence of carcinogenic activity in either the lung or pleura. No treatment-related macroscopic lesions were observed in the lungs or pleura of rats to slag wool at any point of the study. Microscopically, there was a dose- and time-related increase in pulmonary macrophages, microgranuloma formation and bronchiolisation. No evidence of fibrosis at any point. No treatment-related lesions were observed in pleura. No biologically significant adverse health effects observed for the test material. Altogether, repeated dose toxicity studies of Note Q MMVFs tested up to 30 mg/m3 in rats (determined as the MTD) revealed mild and transient effects in the lung such as increased inflammatory responses in alveolar macrophages. No fibrotic potential was seen up to this concentration.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Conclusions:

Testing of repeated dose oral toxicity is waived based on the fact that MMVF note Q fibres are inorganic fibres, whose physicochemical properties suggest a low potential to cross biological membranes and consequently a low potential for absorption through the gastrointestinal tract. MMVF note Q fibres are assessed not to possess toxic properties by repeated ingestion.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study. No deviations.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study included one exposure level of the substance at 30 mg/m3 and a negative control group exposed to filtered air. The exposure duration was 6 hours/day, 5 days/week for 2 years with a subsequent post exposure period lasting approximately until 20% suvival in the filtered air control group.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratory, Raleigh, NC
- Age at study initiation: 7-8 weeks
- Weight at study initiation: approx. 180g
- Fasting period before study: no data
- Housing: individually or in groups of two
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

filtered air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Flow-past exposure chamber. Animals were confined separately in restraint tubes positioned radially at several levels of a vetical aerosol supplytube.
- Method of conditioning air: electron charge neutralization with a Ni-63 line source.
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber and oxygen concetrations were all monitored.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric concentration (mg/m3), WHO Fibers (fibers/cm3) and Fibers L>20µm (fibers/cm3) were monitored throughout the entire exposure period of two years.

Duration of treatment / exposure:

6 hours/day, 5 days/week for 2 years with a subsequent post exposure period lasting approximately until 20% survival in the filtered air control group.

Frequency of treatment:

6 hours/day, 5 days/week for 2 years

Remarks:

Doses / Concentrations:
30 mg/m3
Basis:
other: gravimetric concentration

No. of animals per sex per dose:

140

Details on study design:

- Dose selection rationale: The gravimetric concentration of 30 mg/m3 was selected to obtain a fiber concentration of at least 259 WHO fibers/cm3 throughout the exposure period and for being comparable to the other studies.

Positive control:

No positive control group entered in the study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes: clinical signs, morbidity and mortality
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week during the first 13 wk, then every 2 wk.


NECROPSY was performed on all animals

Sacrifice and pathology:

Scheduled sacrifices after 3, 6, 12, 18 and 24 months of 5 animals per time-point.

Statistics:

Fischer's exact test, one sided.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

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

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEC

Remarks:

(carcinogenicity)

Effect level:

ca. 30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No statistically significant findings of broncho-alveolar hyperplasia, adenomas or carcinomas.

Critical effects observed:

not specified

Summary incidence of broncho-alveolar hyperplasia, bronco-alveolar adenomas, and bronco-alveolar carcinomas in rats at risk for tumour formation:

Group	n	Broncho-alveolar hyperplasia		Adenoma		Carcinoma		Carcinoma + adenoma
		n	%	n	%	n	%	n	%
MMVF note Q fibres	107	6	5.6	5	4.7	0	0.0	5	4.7
Control	107	4	3.7	3	2.8	1	0.9	4	3.7

Note. n, Number of animals "at risk" (defined as the number of animals sacrified at the end of the 12-month exposure period, and the number of animals subsequently found dead or sacrified until the termination of the study, provided that they were exposed for at least 12 months and that their lungs were examined histologically).

Statistical method: Exposed group compared to concurrent control using Fischer's exact test, one-sided. [No statistically significant findings at 5% level.]

Conclusions:

The substance showed minimal collagen deposition in the lungs similar to what could be expected for any biologically inert dust at the same exposure level. It is concluded that the substance does not show any carcinogenic potential in the lungs or pleura.

Executive summary:

This is a comparable to guideline study with no deviations. Furthermore, it was published in a peer-reviewed scientific journal and details on the test materials and experimental design are very well documented. The objective of the study was to assess potential pathogenic and/or oncogenic effects of chronic inhalation exposure to stone wool fibers in rats.

107 male Ficher 344 rats were exposed 6h/day, 5 days/week for 2 years to the substance at 30 mg/m³ by nose-only inhalation of a well-characterized fiber test atmosphere. The study showed that the substance holds neither carcinogenic potential nor any fibrogenic potential in the rat.

Reference 2

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study. No deviations.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Methods of Sachsse et al., (1976); Hesterberg et al., (1991); Bernstein et al., (1993) and Cannon et al., (1983) were followed.

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC.
- Age at study initiation: 6 weeks
- Housing:Polycarbonate cages
- Diet (e.g. ad libitum): Pelleted standard Kliba 343 rat maintenance diet
- Water (e.g. ad libitum): Filtered fresh water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-3 oC
- Humidity (%): 30-70%
- Air changes (per hr): 20 air changes/hr
- Photoperiod (hrs dark / hrs light): 12-hr light/dark

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

Filtered air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Source and rate of air:
- System of generating particulates/aerosols:Research and Consulting Company, Geneva, fiber aerosol generation system.
- Temperature, humidity, pressure in air chamber: 22+-3 oC, 30-70%, -20mm H2O
- Air change rate: 20 air change/hr
- Method of particle size determination: WHO Monograph 4 (WHO, 1985)

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes

During preexposure trials and once every three months thereafter, MMVF note Q fibers at each exposure concentration were captured on filters for determination of fiber length and diameter.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During preexposure trials and once every three months thereafter, MMVF note Q fibres' aerosols at each exposure concentration were captured on filters for determination of fiber length and diameter.

Duration of treatment / exposure:

24 months

Frequency of treatment:

6 hr/day, 5 days/week

Remarks:

Doses / Concentrations:
3, 16, and 30 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

140 (only male rats were used)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: To allow comparison with concurrent studies
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: 6 months
- Section schedule rationale (if not random): Random

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week during the first 13 weeks and at least once a month thereafter.


BODY WEIGHT: Yes
- Time schedule for examinations: Once a week during the first 13 weeks and at least once a month thereafter.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data


CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

Sceduled sacrifices were performed: 3 animals after 3 and 6 moths exposure and 6 animals after 12, 18 and 24 moths exposure.

GROSS PATHOLOGY: No

HISTOPATHOLOGY: Yes
Wagner Pathology Grading Scale was used:
Cellular Change
Normal 1 No lesion
Minimal 2 Macrophage response
Mild 3 Bronchioloization, inflammation, fibrosis
Minimal 4 Minimal
Mild 5 Linking of fibrosis
Moderate 6 Consolidation
Severe 7 Marked fibrosis and consolidation
8 Complete obstruction of most airways

Other examinations:

No data

Statistics:

Pairwise comparison 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. All tests of significance were two-tailed, with no formal adjustment for multiple comparisons. The Student t-test was used for comparisons of the physical characteristics of the fibers.

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 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:

not specified

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

HISTOPATHOLOGY: NON-NEOPLASTIC
There was no evidence of treatment-related interstitial lung fibrosis or pleural fibrosis at any time point in the study. Exposure to the substance was associated with nonspecific inflammatory response (macrophage response) in the lungs that did not appear to progress after 6-12 months of exposure. These cellular changes are reversible and are similar to the effects observed after inhalation of an inert dust. No lung fibrosis was observed.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Exposure resulted in no mesotheliomas and no statistically significant increase in lung tumor incidence when compared to that of negative control group.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant body weight changes or excess mortality during the 2 years of exposure to the substance (data not shown).

Dose descriptor:

NOAEC

Remarks:

lung burden

Effect level:

> 3 - <= 30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Lung burden of the substance after 3-24 months exposure to: 30 mg/m3 (3.72-5.03 fibers/mg dry lung x 10^3), 16 mg/m3 (2.09-3.46 fibers/mg dry lung x 10^3) and 3 mg/m3 (0.35-0.62 fibers/mg dry lung x 10^3), respectively

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Dose descriptor:

NOAEC

Remarks:

(toxicity)

Effect level:

30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEC

Remarks:

(carcinogenicity)

Effect level:

30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No evidence of treatment-related interstitial lung fibrosis or pleural fibrosos. No biologically or statistically significant increase of lung tumor incidence.

Critical effects observed:

not specified

Average Wagner Scores in rats to filtererd air, or MMVF11:

Euthanize/exposure (months)	Air control	MMVF 11
		3 mg/m^3	16 mg/m^3	30 mg/m^3
Continous exposure
3/3	1.0	2.0	2.0	3.0
6/6	1.0	2.0	2.3	3.0
12/12	1.0	2.2	3.0	3.0
18/18	1.0	2.5	3.0	3.0
24/24	1.0	2.5	2.7	2.5
Recovery
24/3	1.0	1.2	2.0	2.0
24/6	1.0	1.5	2.0	2.2
24/12	1.0	2.0	2.0	2.0
24/18	1.0	2.0	2.5	2.2
30/24 c	1.0	2.0	2.2	2.5

c: These animals represent the terminal euthanization which occurred when animal survival was approximately 20%.

Summary of Lung Tumor Findings in Fibrous Glass Study:

Exposure group	At risk b	Adenomas	Carcinomas	Total lung tumors	Mesotheliomas
Controls	123	3 (2.4%)	1 (0.8%)	4 (3.3%)	0
3 mg/m^3	118	3 (2.5%)	1 (0.9%)	4 (3.4%)	0
16 mg/m^3	120	6 (5.0%)	3 (2.5%)	9 (7.5%)	0
30 mg/m^3	112	3 (2.7%)	0	3 (2.7%)	0

b: Only animals that were exposed to fibers for at least 1 year were considered at risk for induction of neoplasms as this was the earliest time point a neoplastic finding was observed in this series of studies.

Conclusions:

Inhalation of MMVF note Q fibres does not have a potential for fibrotic or neoplastic changes in the lungs of rats.

Executive summary:

The study is comparable to a guideline study, and contains all relevant details on the test materials and experimental design. The purpose of the study was to determine the chronic biological effects in Fisher 344 rats of inhaled size-separated respirable fractions of the substance. Rats were exposed either to the substance or to the filtered air (negative control) using nose-only inhalation chambers, 6 hr/day, 5 days/week for 24 months to the concentrations of 3, 16 and 30 mg/m³. Exposure to the substance was associated with a nonspecific inflammatory response in the lungs that did not appear to progress after 6-12 months of exposure. No lung fibrosis was observed in the substance exposed animals, and the exposure resulted in no mesotheliomas and no statistically significant increase in lung tumor incidence.

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

5-day inhalation study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Four different glass wools were evaluated for biopersistence and toxicologic effects following short-term inhalation exposure.
- Short description of test conditions: Rats were exposed nose-only to the test fiber aerosols for 5 days, 6 hours/day and maintained for up to 6 months after exposure.
- Parameters analysed / observed: inflammatory response, lung burden, lung clearance, biopersistence and mortality

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Fiber types used: 901F, 902, JM 901 (MMVF10.1), JM 475 (MMVF33)

The major component of each of the five test fibers is silica.

The glasswools are amorphous (i.e., vitreous). Typical of glasswools, 901F, 902, 901, and 475 are all composed of 57–66% SiO2 and 10–17% Na2O. Compared to the other glasswools, 475 glass is relatively low in CaO, MgO, and Na2O, but relatively higher in ZnO and BaO. Unlike the traditional 901 building insulation, 901F is fluorine free. Compared to the traditional 475, the newer special application fiber, 902, is lower in alumina and higher in the more rapidly dissolving components CaO and Na2O.

JM 901 (MMVF10): 57.40% SiO2, 5.17% Al2O3, 0.072% Fe22O3, 0.03% TiO2, 7.65% CaO, 4.16% MgO, 15.5% Na2O, 1.07% K2O, 0.07% SO3, 8.53% B2O3, 0.68% F2
901F: 57.95% SiO2, 5.00% Al2O3, 0.05% Fe22O3, 0.01% TiO2, 0.02% ZrO2, 0.01% Cr2O3, 6.80% CaO, 4.62% MgO, <0.01% SrO, <0.01% BaO, 16.70% Na2O, 1.08% K2O, 0.06% SO3, 8.48% B2O3
902: 66.44% SiO2, 1.91% Al2O3, 0.054% Fe22O3, 0.041% TiO2, 0.021 ZrO2, 0.002 Cr2O3, 4.63% CaO, 3.18% MgO, 0.003% SrO, 0.17% BaO, 15.95% Na2O, 0.60% K2O, 0.20% SO3, 6.72% B2O3, <0.03% F2
JM475 (MMVF33): 58.63% SiO2, 5.87% Al2O3, 0.043% Fe22O3, 0.012% TiO2, 0.034% ZrO2, 0.004% Cr2O3, 1.74% CaO, 0.24 MgO, 0.11% SrO, 4.98% BaO, 4.02% ZnO, 9.55% Na2O, 3.07% K2O, <0.05% SO3, 11.02% B2O3, 0.62% F2

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: ranged 220-240 g for 901F, 173-220 g for 902 and 901 (biopersistence studies) and 147-188 g for 901F, 475, and positive control amosite (toxicology screening studies)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 0.51 - ca. 0.71 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Fibre aerosol generator and nose-only inhalation exposure system described in separate publication (Bernstein et al., 1995)
- System of generating particulates/aerosols: Fibre aerosol was adjusted to achieve a target concentration of fibres with lengths > 20 µm (F > 20 µm) of at least 100 fibers/cm^3

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test aerosols were monitored for mass, fiber numbers, and bivariate fiber dimensions. Aerosol samples were collected on filters placed in animal exposure ports during testing. Fiber mass concentrations were determined in 2 aerosol samples per day for each of the 5 exposure days. Total fibers were counted in each of 5 daily samples using scanning electron microscopy. Bivariate fiber dimensions measured in 2/5 daily samples according to method outlined by WHO modified for electronic monitoring. Dimensions were measured at on-screen magnification of 7000x in a minimum of 20 fields (approx. 27 µm x 27 µm) for 400 fiber ends (representing measurements of at least 200 fibers). Percentages of fibers in each of several fiber size categories were determined in the 2 daily samples, then used to estimate numbers of fibers in each size category from the total fibers/cc counted in the samples from the remaining 3 days.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days

Dose / conc.:

22 mg/m³ air (analytical)

Remarks:

range 12-32 mg/m³ air
=321-443 WHO fibres/cm³
=0.012-0.032 mg/L

No. of animals per sex per dose:

82-105 male rats exposed to each fibre type, 45-55 rats were exposed to filtered air (the controls)

Control animals:

yes

Details on study design:

Rats were exposed by nose-only inhalation for 5 days/6 hr/day. Post-exposure, rats were maintained for up to 6 months. At 8+ time points during the post-treatment period, 7-15 randomly selected rats/fiber exposure group and 5 control rats were euthanized and evaluated for lung fibre burden (after 1, 2, 3 days; 1 and 2 weeks; 1, 3, 6, months; 45 days (901F only).
Toxicology screening assays were evaluated in 5 rats/group after 1, 10 days, and 1 month of recovery.

Positive control:

Amosite asbestos

Observations and examinations performed and frequency:

Rats were observed daily for mortality and for clinical signs and weighed weekly.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

CLINICAL CHEMISTRY: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes, thoracic histopathology

Other examinations:

Further toxicology screening assays: Pleural-cell proliferation, and pleural and bronchoalveolar lavage (PL and BAL), fluid cytology and clinical chemistry. Each parameter evaluated in 5 rats/exposure group.

Statistics:

Statistical significance of bronchoalveolar and pleural lavage was evaluated by Dunnett test based on pooled variance p < 5%/< 1%.

Clinical signs:

not specified

Mortality:

no mortality observed

Description (incidence):

No mention of fibre-exposed rats that died during or after the 5-day exposure due to severe health effects.

Body weight and weight changes:

not specified

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

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

On recovery day 1, elevations in inflammatory indicators for all fibres were apparent primarily in the bronchoalveolar lavage and only slightly in the pleural lavage. By day 14, elevations in inflammatory indicators were seen in the pleural lavage but not in the bronchoalveolar lavage, for all fibres tested except 901F. On day 29-30, elevations were none/negligible for 901F, 902, and 901, but were striking in bronchoalveolar lavage and pleural lavage for 475 and amosite.
On day 1, all 5 fibres tested induced elevations in several inflammatory parameters, but by one-month post-exposure, symptoms subsided for 901F, 902, and 901, but remained for 475 and amosite.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Exposure-related histopathological effects of glasswools 902 and 901 were only seen on 1 day after the last exposure and limited to fibre-containing microgranulomas in rats exposed to 901 and fibre-containing alveolar macrophages.
After 10 and 30 days, thoracic tissues of exposed rats did not differ from those of the control rats. (901F and 475 not tested in this study). The fibres were associated with initial minimal to slight macrophage aggregation and microgranulomas on recovery day 1.
Wagner scores for 901 and 902 glasswools were normal by 30 days of recovery. Some inflammatory symptoms were associated with 475 and amosite after 30 days; average Wagner scores for animals exposed to 475 and amosite increased slightly.

For individual results see Table 1 in box "Any other information on results incl. tables".

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung burdens: Average lung fibre burdence in the biopersistence studies were 7-12 for WHO fibres and 1.0-1.6 for F >20 µm initial amosite lung burdens tended to be higher than those of the glasswools, especially for total fibres; in millions of total fibres per lung, lung burdens were 10–18 for the glasswools and 23 for amosite.

Details on results:

A lack of lethality or severe health effects after short-term exposure to any of the tested fibres was observed.

Remarks on result:

not determinable because of methodological limitations

Remarks:

Only one concentration tested

Critical effects observed:

not specified

Table 1: Lung histopathology in rats exposed to glasswools or amosite asbestos

Wagner grade: 1 = normal, 2 = macrophage aggregation, 3 = cellularity; 4 = minimal fibrosis, 5 -7 = increasing fibrosis

		Pathology scores
Day after exposure ended	Fibre	Macrophage aggregation	Microgranulomas	Bronchiolization	Wagner grade
Day 1	Air	0	0	0	1
	902	1.2	1.0	0	1
	901	1.0	0	0	1
	901F	1.6	0.8	0	2
Day 14	Air	0	0	0	1
	902	0	0	0	1
	901	0	0	0	1
	901F	0	0.4	0	1.8
	475	0.2	1.8	0	2
	Amosite	1.4	2.0	0	2
Day 29 -30	Air	0	0	0	1
	902	0	0	0	1
	901	0	0	0	1
	901F	0	0	0	1
	475	0.6	1.4	0.4	2.2
	Amosite	0	2.5	1.0	2.6

Conclusions:

No mention of fibre-exposed rats that died during or after the 5-day exposure due to severe health effects. The main effect reported after exposure was increased inflammatory responses in the form of fibre-containing microgranulomas in JM 901-esposed rats and fibre-containing alveolar macrophages in JM 901 or 902-exposed rats seen 1 day after exposure ended. The effects were no longer observed after 10 and 30 days of recovery without fibre exposure (i.e. the effects were reversible).

Executive summary:

In a short-term inhalation study, male Fischer 344 rats (82 to 105 per dose) were exposed to four glass wools (JM 901/MMVF10.1, JM 901F, JM 902, JM 475) and amosite asbestos (positive control) for 5 days, 6 hours/day. The concentration of the fibres ranged between 12 to 32 mg/m³ with the mean concentration of 22 mg/m³. This study demonstrated the lack of lethality or severe health effects after short-term exposure to any of the glass wool fibres. The main effect reported after exposure to the glass wool fibres was increased inflammatory responses in the form of fibre-containing microgranulomas in JM 901-esposed rats and fibre-containing alveolar macrophages in JM 901 or 902-exposed rats seen 1 day after exposure ended. The effects were no longer observed after 10 and 30 days of recovery without fibre exposure (i.e. the effects were reversible).

Reference 4

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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: To assess potential pathogenic and/or oncogenic effects of chronic inhalation exposure to inhaled MMVF34 (HT stone wool) fibres by exposing to rats via inhalation of aerosols and biopersistence of the test material. The publication describes the results of a chronic study (study 1) and an ongoing chronic study (study 2).
- Short description of test conditions: Chronic study 1: Details on the final chronic study are published in McConnell et al. (1994). The main protocol is the same as that described for the ongoing study, except that there are 3 fibre exposure groups (3, 16 and 30 mg/m³) and no specific biopersistence satellite groups were included.
Chronic study 2: Rats are exposed to MMVF34 fibre at 30 mg/m³ for 6 hr/day, 5 days/week for 2 years by nose-only inhalation, with subsequent post-exposure period lasting until approx. 20% survival in the test fibre group.
- Parameters analysed / observed: Adverse effects, necropsy and histopathological findings in relation to fibre exposure and lung-burden data.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Study 1: MMVF34
- stone wool fibre, newly developed commercial insulation wool product
Chemical composition of MMVF34:
- 38.9% SiO2
- 23.2% Al2O3
- 2.1% TiO2
6.7% FeO
15.0% CaO
9.6% MgO
1.9% Na2O
0.8% K2O
0.9% other oxides
in vitro dissolution rates based on Si (ng/cm-2 H-1) of MMVF34: 59 (95% CI 41-77) at pH 7.5; 620 (95% CI 434-806) at pH 4.5

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Albino, SPF Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age at study initiation: 7-8 weeks at delivery
- Weight at study initiation: approx. 180 g at delivery
- Fasting period before study: not specified
- Housing: Animals were housed individual or in pairs in stainless steel wire or polycarbonate cages; optimum hygienic conditions behind a barrier system
- Diet (e.g. ad libitum): pelleted standard rat maintenance diet ad libitum during non-exposure periods
- Water (e.g. ad libitum): tap water, ad libitum

DETAILS OF FOOD AND WATER QUALITY: Water supplied was chlorinated community tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: restraint tubes were positioned radially around the flow-past, nose-only exposure chamber
- Method of holding animals in test chamber: confined separately in restraint tubes
- Source and rate of air: not specified
- Method of conditioning air: not specified
- System of generating particulates/aerosols: Fibre aerosols were produced using the RCC Fibre Aerosol Generation System. This system can produce large concentrations of unbroken fibres with little non-fibrous dust.
- Temperature, humidity, pressure in air chamber: Temperature, relative humidity, and oxygen concentrations were monitored continuously.
- Air flow rate: Airflow rate was monitored daily by constant air pressure for the generation and by flow meter at the chamber extraction.
- Air change rate: not specified
- Method of particle size determination: Bulk fibres were size separated, using a water-based process, to be largely rat respirable and to have a geometric mean diameter of approx. 1 µm and a length of approx. 15 µm.
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: not specified
- Samples taken from breathing zone: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric concentrations and fibre numbers were monitored at regular intervals during the studies.

Duration of treatment / exposure:

2 years

Frequency of treatment:

6 hr/day; 5 days/week

Dose / conc.:

30 mg/m³ air (nominal)

No. of animals per sex per dose:

5 animals per exposure time (3, 6, 12, 18, 24 months)

Control animals:

yes, concurrent vehicle

Positive control:

MMVF21 (less biosoluble MMVF than MMVF34)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: clinical signs, morbidity, mortality
Individually examination was performed each week during the first 13 weeks, then every second weeks

BODY WEIGHT: Yes
- Time schedule for examinations: 1x week during first 13 weeks, then every second week

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Lungs removed, weighed and examined under a dissecting microscope
HISTOPATHOLOGY: Yes
- Performed on all animals from the 3, 6, 12, 18-month sacrifices.
- Lungs stained with Trichrome (Masson-Goldner Method) and with Haematoxylin and Eosin
- Lungs were examined and classified histopathologically and given a Wagner score for inflammatory change and fibrosis. [Wagner score: cellular change: 1 = normal; 2 = minimal; 3 = mild; 4 = minimal; 5 = mild; 6 = moderate; 7-8 = severe.
Fibrosis: 4 = minimal; 5 = mild; 6 = moderate; 7-8 = severe]

Other examinations:

Lung burden analysis:
- At necropsy, lung lobes from each animal were removed (from animals in chronic study with histopathology only accessory and right caudal lobe), weighed and frozen. Post-thaw, lobes were finely minced with scissors, dried to a constant weight and the lungs digested using low-temperature ashing. The qualification and quantification of fibre retention relied on lung-burden analyses using SEM.
- Approx. 200-400 fibres per lung sample was analysed. In each group, fibre count for evaluation of lung burden and bivariate analysis of size for evaluation of size distribution were performed on ashed lungs of the animals at each scheduled sacrifice time-point.

Statistics:

Using lung burden data, kinetics were calculated of the elimination of the fibres from the lung by regression analysis of the logarithm of the number of fibres versus time after termination of exposure, corresponding to single exponential model (Fraunhofer-ITA 1997)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

MMVF34: Clinical signs were recorded in isolated animals, not attributed to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

MMVF34: Body weight gains were comparable between the exposed and the air-control groups, but the mean body weight in the control group sacrificed at 3 months was slightly higher than in the exposed group. Potentially related to some discomfort associated with the initial administration of fibres.

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the 3, 6, 12, and 18 months sacrifices, the mean lung weights in the MMVF34 group showed a statistically significant increase compared to control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MMVF34 showed less evidence of pulmonary fibrosis than animals exposed to MMVF21, which itself demonstrated only low levels of pathogenicity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Throughout the 18-month period, only slight macrophage reaction was seen in lungs of rats exposed to MMVF34 (Wagner grade 2). In some animals, occasional micro-granulomas or small areas of bronchiolisation seen at the bifurcations of the terminal or respiratory bronchioles (Wagner grade 3).

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

MMVF34: No information available on development of tumours

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung burden: For MMVF34 long fibres (L>20 µm), a steady state lung burden has apparently been reached by 3 to 6 months exposure.

Details on results:

The carcinogenicity and toxicity results of the chronic study with the positive control MMVF21 suggest that this fibre does not pose a significant health risk to humans and the current results with MMVF34 indicate that this fibre consequently poses an even smaller risk, if any. For MMVF34 throughout the 18 month period, only slight macrophage reaction was seen in the lungs of rats exposed to MMVF34 (Wagner grade 2) although in some animals occasional microgranulomas or small areas of bronchiolization were seen at the bifurcations of the terminal or respiratory bronchioles (Wagner grade 3).

For individual results on lung burdens and pulmonary changes see Table 1 in box "Any other information on results incl. tables".

Remarks on result:

not determinable because of methodological limitations

Remarks:

One concentration used for testing Note Q MMVF

Critical effects observed:

not specified

Table 1: Lung burdens per mg dry lung and pulmonary changes (mean Wagner scores) after different periods of exposure

	Exposure aerosol		Lung burden (Fibres per mg dry lung x 10³)				Interstitial fibrosis (Mean Wagner Score)
Fibre	mg/m³	L > 20 µm/cm³ WHO/cm³	3 months exposure L > 20 µm WHO	6 months exposure L > 20 µm WHO	12 months exposure L > 20 µm WHO	18 months exposure L > 20 µm WHO	3 months exposure	6 months exposure	12 months exposure	18 months exposure
MMVF 21	16.1	74	8	16	37	58	2.2	2.7	2.7	4.0
		150	38	85	210	233
	30.4	114	18	23	55	62	3.2	3.3	3.3	4.0
		243	83	143	319	283
MMVF34	30.5	86	8	11	10	11	1.6	2.6	2.6	2.8
		288	108	147	152	222

Conclusions:

The carcinogenicity and toxicity results of the chronic study with the "positive control" MMVF21 suggest that this fibre does not pose a significant health risk to humans and the current results with MMVF34 indicate that this fibre consequently poses an even smaller risk, if any.

Executive summary:

In a subchronic inhalation study, the two stone wool fibre types MMVF34 (newly developed commercial insulation wool product) and MMVF21 (traditional stone wool) were administered to male Fischer 344/N rats (5 to 6 animals per exposure time) for 6 hr/day; 5 days/week up to two years. Adverse effects, necropsy and histopathological findings in relation to fibre exposure and lung-burden data were examined. In this study, MMVF21 produced a dose-related non-specific inflammatory response and minimal focal pulmonary fibrosis late in the exposure period but no evidence of carcinogenic activity in either the lungs or the pleura. An observed pulmonary fibrosis, in addition to an increase in lung weights, indicates that the maximum tolerated dose had been achieved in this study. Only preliminary histopathological results up to 18 months exposure are available from the chronic inhalation study with MMVF34 but the histopathological changes are minor compared to MMVF21 and based on the available results it seems unlikely that MMVF34 exposed animals will develop pulmonary tumors in excess of those that develop in the control. When comparing the pathology after 3, 6, 12 and 18 months exposure, MMVF34 showed minor histopathological changes compared to MMVF21. The carcinogenicity and toxicity results of the chronic study with MMVF21 suggest that this fibre does not pose a significant health risk to humans, and the current results with MMVF34 indicate that this fibre consequently should pose an even smaller risk, if any.

Reference 5

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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: Acute inhalation toxicity test using rats
- Short description of test conditions: Rats were exposed nose-only to two stone/rock wool fibres (MMVF34 and MMVF21) in aerosol for 5 days, 6 hr/day.
- Parameters analysed / observed: Biopersistence, morbidity and mortality

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test material used: MMVF34

Chemical composition of MMVF34:
- 38.9% SiO2
- 23.2% Al2O3
- 2.1% TiO2
6.7% FeO
15.0% CaO
9.6% MgO
1.9% Na2O
0.8% K2O
0.9% other oxides
in vitro dissolution rates based on Si (ng/cm-2 H-1) of MMVF34: 59 (95% CI 41-77) at pH 7.5; 620 (95% CI 434-806) at pH 4.5

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Albino, SPF Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age at study initiation: 7-8 weeks at delivery
- Weight at study initiation: approx. 180 g at delivery
- Fasting period before study: not specified
- Housing: Animals were housed individual or in pairs in stainless steel wire or polycarbonate cages; optimum hygienic conditions behind a barrier system
- Diet (e.g. ad libitum): pelleted standard rat maintenance diet ad libitum during non-exposure periods
- Water (e.g. ad libitum): tap water, ad libitum

DETAILS OF FOOD AND WATER QUALITY: Water supplied was chlorinated community tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: restraint tubes were positioned radially around the flow-past, nose-only exposure chamber
- Method of holding animals in test chamber: confined separately in restraint tubes
- Source and rate of air: not specified
- Method of conditioning air: not specified
- System of generating particulates/aerosols: Fibre aerosols were produced using the RCC Fibre Aerosol Generation System. This system can produce large concentrations of unbroken fibres with little non-fibrous dust.
- Temperature, humidity, pressure in air chamber: Temperature, relative humidity, and oxygen concentrations were monitored continuously.
- Air flow rate: Airflow rate was monitored daily by constant air pressure for the generation and by flow meter at the chamber extraction.
- Air change rate: not specified
- Method of particle size determination: Bulk fibres were size separated, using a water-based process, to be largely rat respirable and to have a geometric mean diameter of approx. 1 µm and a length of approx. 15 µm.
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: not specified
- Samples taken from breathing zone: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric concentrations and fibre numbers were monitored at regular intervals during the studies.

Replicate fibre samples were subjected to a continuous constant flow (Flow-rate/Initial Surface Area = 0.03 µm/s) of buffer solution (modified Gamble's solution) at two different pH levels in a dissolution rate measurement apparatus. Dissolution rates were calculated on the basis of the analysis of dissolved Si.
The gravimetric concentration was adjusted to obtain a concentration of long fibres (L >20 µm) of 150 fibres/cm³ in order to increase lung burdens and to increase statistical confidence in the results.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hr/day

Dose / conc.:

60 mg/m³ air (analytical)

No. of animals per sex per dose:

66 males

Control animals:

yes, concurrent vehicle

Positive control:

MMVF21 (traditional stone wool fiber)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: clinical signs, morbidity, mortality
Individually examination was performed each week during the first 13 weeks, then every second weeks

BODY WEIGHT: Yes
- Time schedule for examinations: 1x week during first 13 weeks, then every second week

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No

Other examinations:

Lung burden analysis:
- At necropsy, lung lobes from each animal were removed (from animals in chronic study with histopathology only accessory and right caudal lobe), weighed and frozen. Post-thaw, lobes were finely minced with scissors, dried to a constant weight and the lungs digested using low-temperature ashing. The qualification and quantification of fibre retention relied on lung-burden analyses using SEM.
- Approx. 200-400 fibres per lung sample was analysed. In each group, fibre count for evaluation of lung burden and bivariate analysis of size for evaluation of size distribution were performed on ashed lungs of the animals at each scheduled sacrifice time-point.

Statistics:

Using lung burden data, kinetics were calculated of the elimination of the fibres from the lung by regression analysis of the logarithm of the number of fibres versus time after termination of exposure, corresponding to single exponential model (Fraunhofer-ITA 1997)

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse symptoms observed

Mortality:

no mortality observed

Description (incidence):

No adverse symptoms observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse symptoms were 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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Lung burden: The biopersistence results after short-term inhalation exposure showed a rapid removal of MMVF34 long fibres (L > 20 mm) from the lungs compared to MMVF21.

Remarks on result:

not determinable because of methodological limitations

Remarks:

One concentration tested

Critical effects observed:

not specified

Conclusions:

In this study, no effects on morbidity and mortality was observed in rats after 5 days exposure. The biopersistence results after short-term inhalation exposure showed a rapid removal of MMVF34 long fibres (L > 20 mm) from the lungs compared to MMVF21.

Executive summary:

In a subacute inhalation study, the Note Q stone wool fibre type MMVF34 (newly developed commercial insulation wool product) was administered to male Fischer 344/N rats (5 to 6 animals per exposure time) at a dose of 60 mg/m³ in air for 6 hr/day; 5 days/week with subsequent post-exposure periods lasting up to 12 months. Morbidity, mortality and lung burden was examined.

Adverse effects, necropsy and histopathological findings in relation to fibre exposure and lung-burden data were examined. The traditional non-Note Q fibre MMVF21 served as a positive control.

 

No effects on morbidity and mortality was observed in rats after 5 days exposure. The biopersistence results after short-term inhalation exposure showed a rapid removal of MMVF34 long fibres (L > 20 mm) from the lungs compared to MMVF21.

Reference 6

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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: Subchronic inhalation study in rats to evaluate the effects of new biosoluble high-aluminium low silica HT type stone wool. The study was conducted according to the EU protoxol for conducting subchronic inhalation studies in Man-made vitreous fibres (European Joint Research Centre, 1999)
- Short description of test conditions: Male Wistar rats exposed nose-only to 15 mg/m³ aerosol stone/rock wool (MMVF21) and 3 stone wool fibres (RIF41001, RIF42020-6; length >20 µm) for 6 hr/day, 5 days/week for 3 months
- Parameters analysed / observed: Pulmonary change and fibre numbers in lung, including bronchoalveolar lavage fluid for evaluation of inflammatory response and measurement of cell proliferation, assessment of early fibrosis through histological examination and comparison of body weight and lung lobe weights.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

HT wool (high-aluminium low-silica): CAS-No. 287922-11-6
RIF41001: 42.7% SiO2, 18.9% Al2O3, 1.6% TiO2, 6.3% FeO, 18.3% CaO, 7.8% MgO, 1.7% Na2O, 0.9% K2O, 0.2% other oxides
RIF42020-6: 36.8% SiO2, 20.7% Al2O3, 1.4% TiO, 5.4% FeO, 18.0% Cao, 10.2% MgO, 4.4% Na2O, 0.5% K2O, 0.9% other oxides

According to the study by Guldberg et al. (2000), the composition of the stone wool fulfill the criteria for exoneration from classification as a possible carcinogen according to the European Commission Directive from 1997 and/or the German regulation (TRGS 905, 1999) based on the short-term in vivo biopersistence tests.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats recommended by EPA workshop (Vu et al. 1996) for use in subchronic and chronic inhalation toxicity studies of fibres. According to EU guidelines, the use of male Wistar rats is preferred.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 200-300 g
- Housing: Animals were housed in groups of two in polycarbonate cages when not being exposed; optimum hygienic conditions behind barrier system
- Diet (e.g. ad libitum): Pelleted standard rat maintenance diet; ad libitum
- Water (e.g. ad libitum): Tap water; ad libitum
- Acclimation period: 2 weeks, and accustomed to nose-only tubes for 3 weeks pre-exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 40-70%
- Air changes (per hr): air conditioned; not specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 0.54 - <= 0.62 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only, flow-past technique; fiber aerosol is supplied to each animal individually and exhaled air is immediately exhausted.
- Method of holding animals in test chamber: Battelle type polycarbonate tubes; animals keep noses close to airflow at opening of tube. Exposure of animals was performed in identical exposure chambers of cylindrical shape, each housing up to 48 animals.
- Method of conditioning air: airflow, temperature, humidity monitored continuously and were stored as 20 min mean values.
- System of generating particulates/aerosols: High pressure pneumatic disperser, fed with test substances under computerized control, with feedback to the actual aerosol concentrations measured by an aerosol photometer. Aerosols were neutralized by a 63-Ni source to reduce the charge on the fibres.
- Temperature, humidity, pressure in air chamber: Airflow
- Air flow rate: approx. 1 L/min (laminar)
- Method of particle size determination: Lengths and diameters performed using SEM at magnification of at least 2000. Recording of particles was stopped when a total of 30 particles had been counted.

TEST ATMOSPHERE
- Brief description of analytical method used: not necessary to measure oxygen concentration because environment was laminar
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual aerosol concentrations were measured by an aerosol photometer. The photometer gives a scatter light signal, which is nearly proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the studyby comparison with gravimetric and fibre concentration. The aerosols of the test substances were neutralised by a 63Ni source to reduce the charge on the fibres.

Duration of treatment / exposure:

3 months

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

15 mg/m³ air (nominal)

Remarks:

RIF41001: 15.9 mh/m³
RIF42020-6: 14.6 mg/m³

No. of animals per sex per dose:

32 male rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Animals randomized to treatment groups stratified on body weight. 32 rats (including 2 reserve animals) per exposure group. 10 rats allocated for each post-exposure kill group. 5 animals killed at each time point for histopathology also used for the lung burden determinations.

Positive control:

MMVF21

Observations and examinations performed and frequency:

Rats examined daily for clinical signs, morbidity, and mortality. Individually examined outside the cage once a week. Body weight recorded once per week during the first 3 months, then every second week. Lavagate was used to determine biochemical parameters (LDH, beta-glucuronidase and total protein).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

At the scheduled kills the lungs were removed in toto, weighed without the trachea and carefully examined. The right lobes were removed, weighed and deep frozen for lung fibre burden analysis (see below). The left lung lobe was perfused with fixative via the trachea at a pressure of 20 cm water for 2 h.

Other examinations:

Assessments: bronchoalveolar lavage fluid (BALF) for evaluation of inflammatory response (e.g. protein content, enzymes, increase in polymorphnuclear leucocytes) and measurement of cell proliferation, assessment of early fibrosis through histological examination and comparison of body weight and lung lobe weights.

Statistics:

Individual toxicological data were compared to clean air control group. For numeric parameters, the statistics were done using Dunnett's test. For comparison of pathological findings, a pairwise Fischer's test between control and treatment groups was performed.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs recorded in isolated animals, not attributed to treatment with test material.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality was recorded in isolated animals, not attributed to treatment with test material.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gains comparable between exposed and air control group.

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:

effects observed, treatment-related

Description (incidence and severity):

LDH and total protein significantly elevated in all groups at end of exposure kill and remained elevated 3-months post-exposure.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 3 months exposure, mean lung weights in HT and MMVF21 exposed groups were statistically significantly increased compared with control. At the 3-month post-exposure sacrifice, the HT group no longer had significant increase in lung weight, but the MMVF21 did.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Lung fibre burden:
In both stock fibres and aerosol fibres the diameter of the HT fibres was smaller than that of the MMVF21 fibres. However, when the lung fibre burden was examined at the first-time point, the dimension of the retained fibres was very similar for all fibre types. This probably indicates that the lung filters out the thicker fibres, so that the lung tissue is exposed to very similar fibre dimensions regardless of the fibre type. The maximum lung burden achieved was considerably lower in the HT groups than in the MMVF21 group, except in the RIF41001 group, which showed comparable numbers at the end of exposure. Even with this HT type the long fibres cleared much faster in the recovery period than MMVF21.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

All control animals graded 1 (according to Wagner scale). Rats exposed to HT fiber types had lower Wagner scores than animals exposed to MMVF21. Only slight macrophage reaction seen in lungs of rats exposed to HT fibre types (Wagner 1-2). At 3-months' exposure, Wagner grade 1 noted in all animals exposed to RIF41001. In rats exposed to RIF42020-6, Wagner grade 1 was noted in 2 rats and grade 2 noted in 3 rats. Wagner grade 2 characterized by focal minimal microgranulomas at the bronchiolar-alveolar junction. The MMVF21 group had 1 rat with grade 3, 4 rats with grade 4, measured at 3 months' exposure. After 3 months' exposure, differences between HT and MMVF21 were seen for all the histological parameters examined (alveolar bronchiolization, microgranulomas, collagen deposition at bronchiolar-alveolar junction, pleural collagen deposition, macrophages in alveolar lumina). In control group, there were 0 lesions.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Cell proliferation enhanced at end of exposure for MMVF21 for all three labelling indices, but only for the bronchiolar epithelium in RIF41001 group. No significant increase in cell proliferation found at kill dates after the 3-month exposure ended.

Details on results:

For HT and MMVF21, a significant increase in PMN and reduction in percentage of macrophages in bronchoalveolar lavage fluid was observed.
No clear difference in bronchoalveolar lavage fluid cell concentration and percentage of cells between MMVF21 and HT groups, though the percentage of PMNs in the HT groups tended to be higher.

Remarks on result:

not determinable because of methodological limitations

Remarks:

Only one concentration tested

Critical effects observed:

not specified

Conclusions:

In this study, mild pathological changes were found in the lungs of rats exposed with HT. Further changes included alveolar macrophage aggregation and/or microgranulomas at the bronchiol alveolar junction in the few rats affected. No fibrogenic potential was noted of the two HT fibres.

Executive summary:

In a subchronic inhalation study, male Wistar rats (32 per dose) were exposed nose-only to new biosoluble high-aluminum low-silica HT type stone wool (RIF41001 and RIF42020 -6) in air for 6 hours/day, 5 days/week for 3 months, and followed post-exposure for several months. The parameters measured included pulmonary change and fibre numbers in lung, including bronchoalveolar lavage fluid for evaluation of inflammatory response and measurement of cell proliferation, assessment of early fibrosis through histological examination and comparison of body weight and lung lobe weights. After exposure of rats to the new biosoluble fibers (HT), no biologically significant effects were observed except that a statistically significant increase in lung weight was observed up to 1.5 months post-exposure in all 3 treatment groups. At 3 months post-exposure, the small increase was no longer significant. Minimal morphological changes were diagnosed in the HT fibre groups at 3 months and 1.5 and 3 months post-exposure. No fibrogenic potential noted of the two HT fibres. No clear-cut difference between the different biosoluble fibre types noted. Only slight macrophage reaction seen in lungs of rats exposed to HT fibre types: Wagner grade 1 or 2 in all animals examined.

Reference 7

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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: Chronic nose-only inhalation study in Fischer 344/N rats to investigate the potential pathogenic effects of two different types of man-made vitreous fibres.
- Short description of test conditions: Male rats were exposed nose-only to rock wool and slag wool in air for 6 hours/day, 5 days/week for 24 months. Rats were held post-exposure for lifetime observation (until approx. 20% survived).
- Parameters analysed / observed: Clinical signs, morbidity, mortality, necropsy, lung fiber burden measurements, progression of pulmonary lesions during exposure period and post-exposure period.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Slag wool:
- Source: USG Interiors (USA) slag wool
Slag wool chemical composition by weight percent: 38.4% SiO2, 10.6% Al2O3, 38.0% CaO, 9.9% MgO, 0.3% Fe2O3, 0.4% Na2O, 0.5% K2O, 0.5% TiO2, 0.7% MnO, 1.8% S

Other details: Bulk fibers with chemical compositions typical of commercial insulation products were size separated to be largely rat respirable (<1.5 µm diameter and <80 µm length), and to have average dimensions (average aerosol geometric mean diameter <1.0 µm and length >15 µm). Approx. 4000 kg of bulk product was size separated using a water-based process to obtain 10 kg of stock fiber with the required dimensions to be used in the inhalation study.

Species:

rat

Strain:

Fischer 344

Remarks:

344/N

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: 6 weeks old
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: Animals were housed individually in polycarbonate cages containing hardwood bedding in Hazelton 2000 chambers under negative pressure (-20 mm H20)
- Diet (e.g. ad libitum): pelleted feed (Kliba 343, Klingentalmuehle AG, Switzerland); ad libitum
- Water (e.g. ad libitum): filtered fresh water; ad libitum
- Acclimation period: 2 week quarantine period

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

not specified

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Rats placed in exposure tubes
- Method of holding animals in test chamber: confined separtaely in tubes positoined radially around the exposure chamber
- Source and rate of air: Flow-past nose-only sytem 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.
- Method of conditioning air: not specified
- System of generating particulates/aerosols: A system of nondestructive aerosolization to to maximize the number of "rat-respirable fibers". It produces large numbers of unbroken fibers with lower levels of nonfibrous dust.
- Temperature, humidity, pressure in air chamber: levels not specified, but monitored daily
- Method of particle size determination: Fiber mass concentrations were measured at least 4x/week during the 2-year exposure period. Each aerosol sample from each fiber type and dose level was collected. The aerosols were analyzed for WHO fibers per cubic centimeter

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Fiber mass concentrations were measured at least 4 times/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 Gellman membrane filter from one of the laminar flow ports int eh exposure chamber to elimniate isokinetic sampling bias. Aerosol concentrations were monitored continuously during the exposure period using an RAS light-scattering monitor to assure uniformity. The aerosols were analyzed for WHO fibers per cubic centimeter once each exposure week during week 1-13 and every other week thereafter through exposure week 104. The total numbers of fibers and non-fibrous particles were counted once aech month on a filter for each fiber type and dose. Samples were collected on Millipore filters, placed between glass slides, clarified, counted by phase-contrast microscopy at 400x magnification. At least 20 fields counted on each slide. Once per month, total fibers and particles were counted to determine total fibers per cubic centimeter and particles per cubic centimeter.
Every 3 months, samples of slag wool aerosols for each dose were collected on filters for determination of fiber length and diameter.

Duration of treatment / exposure:

24 months

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

3 mg/m³ air (nominal)

Remarks:

gravimetric concentration 3.06 ± 0.44 mg/m³

Dose / conc.:

16 mg/m³ air (nominal)

Remarks:

gravimetric concentration 16.11 ± 1.39 mg/m³

Dose / conc.:

30 mg/m³ air (nominal)

Remarks:

gravimetric concentration 29.89 ± 2.87 mg/m³

No. of animals per sex per dose:

140 rats per exposure group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose (30 mg/m³) was chosen to be comparable to other studies of MMVFs
There were 140 rats in each rock and slag wool exposure group, positive control, unexposed chamber control groups exposed for 6 hr/day, 5 days/week for up to 24 months.
Groups of 6 randomly selected rats, identified at the start of the study, from each exposure group were killed at 3, 6, 12, 18, 24 months for lung fibre burden measurements and to follow progression of pulmonary lesions. In addition, groups of 3-6 rats from each group were removed from exposure at 3, 6, 12, 18 months and held until 24 months, at which time they were sacrificed to ascertain whether there was progression or regression of lesions and to determine lung fibre burdens following cessation of exposure.

Positive control:

Crocidolite asbestos (10 mg/m³)

Observations and examinations performed and frequency:

Test animals were observed daily for cilnical 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 each month after.

Sacrifice and pathology:

At necropsy, the accessory lobe of the lung was removed, weighed, frozen, then thawed, dehydrated, and evaporated to constant weight. The fibers were counted and measured similar to the way the aerosol fiber samples were counted. Other tissues were processed routinely including nasal cavity, larynx, trachea, bronchi, mediastinal, mesenteric lymph nodes, liver, spleen, kidneys, heart, an dall grossly visible lesions. Lungs were examined and classified histopathologically and given a Wagner score for inflammatory change and fibrosis: 1 = normal, 2 = minimal (macrophage response), 3 = mild (macrophage/bronchiolization), 4-8 denote degrees of fibrosis, 4 = minimal (fibrosis restricted to terminal bronchioles/proximal alveoli), 5 = mild (interlobular linking), 6 = moderate (early consolidation), 7 = severe (marked fibrosis/consolidation, 8 = complete obstruction of airways.

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 months (crocidolite for 10 months) were considered at risk for induction of neoplasms. All tests of signficance were two-tailed, with no formal adjustment for multiple comparisons. Student's t-test was used for comparisons of teh physical characteristics of the fibers.

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal clinical signs were observed.

Mortality:

no mortality observed

Description (incidence):

Survival was comparable to unexposed controls.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain was comparable to unexposed controls.

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

Immunological findings:

not examined

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 any of the slag-wool-exposed rats. No lung weight differences observed at 24 months in any of the groups compared to controls, probably due to high incidence of pulmonary leukemia, which is common in older F344/N rats.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic lesions were observed in the lungs or pleura of rats exposed to slag wool at any point during the study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dose- and time-related increase in pulmonary macrophages, microgranuloma formation, and bronchiolization seen. Fibres and fibre fragments found in many macrophages and microgranulomas. However, there was no evidence of fibrosis at any time. No treatment-related lesions were observed in the pleura.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Slag wool: Occasional bronchoalveolar neoplasms were found: 2/116 low dose, 0/115 mid dose, 3/115 high dose, compared to 2/126 unexposed controls. No treatment-related lesions were observed in upper respiratory track or in any other organ. Many rats, including unexposed controls, showed evidence of mononuclear cell leukemia involvement of lung (diffuse gray discoloration, paleness, and/or multifocal punctuate gray foci) at 24 months accompanied by enlarged spleen and nodular liver.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung burden
The average lung burdens of WHO fibres reached their peak at 1 yr and plateaued during the remainder of the study. The retention of slag wool fibres decreased rapidly after exposure ceased. The decrease was mire dramatic for fibres > 20 µm in length for slag wool, which decreased from 31 000 to 200. The electron microscopic examination used in the counting of the fibres also revealed ultrastructural changes in the MMVF fibres after retention in the lung. After 6 months the slag wool fibres showed segmental dissolution and cross cleavage. In contrast, the crocidolite fibres showed no morphologic change, even after 24 months in the lung.

Dose descriptor:

NOAEC

Effect level:

30 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Critical effects observed:

not specified

Exposure atmosphere and fibre characterisation

The three airborne gravimetric concentrations (mg/m³ ± SD) of rock wool averaged 3.06 ± 0.41, 16.14 ± 1.17, and 30.39 ± 2.79 and slag wool 3.06 ± 0.44, 16.11 ± 1.39, and 29.89 ± 2.87 during the 2 year exposure. These concentrations corresponded to average WHO fibre numbers (fibres/cm³ ± SD) of 34 ± 13, 150 ± 51, and 243 ± 67 for rock, and 30 ± 15, 131 ± 40, and 213 ± 62 for slag. The average numbers of rock wool fibres > 10 µm in length were 26 ± 9, 122 ± 33, adn 186 ± 51 and those > 20 µm were 13 ± 5, 74 ± 20, and 114 ± 32. Slag wool fibres > 10 µm in length numbered 20 ± 8, 96 ± 28, and 165 ± 51 and those > 20 µm were 10 ± 4, 50 ± 14, and 99 ± 31. The aerosols also contained nonfibrous particulates. The crocidolite asbestos aerosol (positive control) was 10.04 ± 0.90 mg/m³ which corresponded to an average of 1610 ± 989 WHO fibres/cm³. The average number of fibres/cm³ > 10 µm in length was 677 ± 416, while the number of those > 20 µm was 236 ± 145.

Conclusions:

Non-specific inflammatory response, no evidence of carcinogenic activity in either the lung or pleura. No treatment-related macroscopic lesions were observed in the lungs or pleura of rats to slag wool at any point of the study. Microscopically, there was a dose- and time-related increase in pulmonary macrophages, microgranuloma formation and bronchiolisation. No evidence of fibrosis at any point. No treatment-related lesions were observed in pleura. No biologically significant adverse health effects observed for the test material MMVF22.

Executive summary:

In a chronic inhalation study, male Fischer 344/N rats (160 per dose) were exposed nose-only to slag wool MMVF22 (a Note Q MMVF) in air for 6 hours/day, 5 days/week for 24 months. Rats were held post-exposure for lifetime observation (until approx. 20% survived). Crocidolite asbestos (10 mg/m³) was used as a positive control. Clinical signs, morbidity, mortality, necropsy, lung fibre burden measurements and progression of pulmonary lesions during exposure period and post-exposure period were investigated. No abnormal clinical signs, changes in body weight or mortality were observed. Non-specific inflammatory response, no evidence of carcinogenic activity in either the lung or pleura. No treatment-related macroscopic lesions were observed in the lungs or pleura of rats to slag wool at any point of the study. Microscopically, there was a dose- and time-related increase in pulmonary macrophages, microgranuloma formation and bronchiolisation. No evidence of fibrosis at any point. No treatment-related lesions were observed in pleura. No biologically significant adverse health effects observed for the test material MMVF22.

Reference 8

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

5 days exposure

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Short-term inhalation study in rats to determine biopersistence of the test material by analyzing the concentrations in lung tissue
- Short description of test conditions: Rats were exposed nose-only to 30 mg/m³ of two fiber glass compositions (rockwool and slagwool) with filtered air as a control 6 h/day for 5 days. Animals were sacrificed at various time intervals ranging from 1 hour to 545 days post-exposure and fibers were recovered from digested lung tissue to determine changes in concentrations and fiber retention.
- Parameters analysed / observed: Digested lung tissue analyzed to determine changes in concentrations (fibers/mg dry lung) and fiber retentions (expressed as percent of day 1 retention [PR]) for selected dimension categories.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Glasswool:
- Fiber 10, source: Manville Corp.
- Fiber 11, source: Certainteed Corp.

Rockwool:
- Fiber 21, source: Roxul International

Slagwool:
- Fiber 22, source: USG Interiors

From each MMVF bulk product, the fraction selected had fibers of average dimensions of approximately 1 µm diameter and 15 µm length. Fibers of such dimensions are found in workplace aerosols. The fibers obtained accounted for 0.25 to 5 wt-% of the bulk product, depending on the initial diameter distribution of the MMVF.

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

no data

Sex:

not specified

Details on test animals or test system and environmental conditions:

no data

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: filtered air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Fiber aerosols were lofted using a generation system designed not to break, grind, or contaminate the fibers
- Method of particle size determination: optical and scanning electron microscopy was used for length and diameter dimensions. Diameters were measured at x5000 magnification, from a
minimum of 200 fields or 100 counted fibers. Length measurements were made by optical microscopy at x1500 magnification to reduce the risk of truncated fibers.

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol concentrations for fiber mass were monitored by gravimetric sampling.

- Samples taken from breathing zone: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Fiber concentration levels (fibers/cm^3) were determined by phase contrast microscopy (x400 magnification) for total and WHO fiber numbers.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hr/day

Dose / conc.:

30 mg/m³ air

Remarks:

gravimetric concentration

No. of animals per sex per dose:

9 animals per dose (sex not specified)

Control animals:

yes

Details on study design:

Groups of 9 rats were exposed "nose-only" for 6 hr/day for 5 days to one concentration of 30 mg/m3 for each MMVF. Another group was exposed to 10 mg/m3 of crocidolite fibers and another group breated filtered air.

Positive control:

no data

Observations and examinations performed and frequency:

Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: No

Other examinations:

Tissue concentrations:
9 rats for each fiber type and 9 controls were sacrificed at 1 hour, 1, 5, 31, 90, 180, 270, 365, 545 days after fiber inhalation ended. Lungs were removed, infracardiac lobes separated, and remaining lung tissues preserved. Lobes from 5 rats from each exposed group and from the controls were dehydrated and dried to constant weight to obtain the initial dry weight of tissue for determination of fiber concentrations. Tissues were low temperature ashed, fibers recovered, fiber number, diameters and lengths were determined via SEM and optical microscopy.

Statistics:

Concentrations of fibers per mg of dry lung tissue (f/mg DL) were calculated, as were concentrations of fibers and the percentages of day 1 retention (or PR) for selected diameter and length categories for each sacrifice time point.

Clinical signs:

not examined

Mortality:

no mortality observed

Body weight and weight changes:

not examined

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

See box "Any other information on results incl. tables".

Details on results:

Results from this study indicated that, of the long crocidolite fibers retained in the pulmonary region at day 1, approximately 40% remained through 545 days.
The nearly complete disappearances of MMVF fibers ≥0.5 µm in diameter and >20 µm in length within 270 days postexposure strongly suggested dissolution. These disappearances were accompanied by increases in the percentages observed for fibers <0.5 µm in diameter and <5 µm in length, suggesting that the thicker and longer fibers had dissolved or broken with time, "enriching" the thinner and shorter fractions. In addition, none, or very few MMVF fibers ≥20 µm in length were detected in the lung tissues from exposed animals at 545 days, whereas for crocidolite, fiber concentration at 545 days remained at 2000 ± 400 f/mg DL.

Remarks on result:

not determinable because of methodological limitations

Remarks:

Only one concentration tested

Critical effects observed:

not specified

Geometric mean diameters

The average diameters and lengths of crocidolite fibers recovered from the rat lungs at various retention times differed little, whereas the MMVF showed a decrease in both geometric mean diameter and length with time retained in the animals' lungs.

Fiber concentrations in lung

Fibers recovered from the 1-hr sacrifice represent the deposition in both the conducting airway and alveolar regions of the lung, since particle clearance from the conducting airways is assumed to be complete within 24 hr after stopping exposure. For crocidolite fibers ≥5 µm in length, the average fiber concentrations from the day 1 sacrifice were 168,000 ± 47,000 and for 31, 100,000 ± 31,000 f/mg DL. The average decreased to 58,000 ± 12,000 at day 365 sacrifice and 56,000 ± 6000 at day 545.

Long crocidolite fiber retention tended to be prolonged compared to retention of crocidolite particles (i.e., <5 µm long) as indicated by higher retention values for the longer fibers beginning after 31 days. Percent retentions of fibers≥10 µm did not differ significantly from the PRs of fibers ≥5 µm. PRs of fibers >20 µm in length tended to be higher than those for fibers ≥5 µm at 365 and 545 days. For crocidolite fibers >20 µm in length, average concentrations were roughly equal between days 1 and 31 (first day, 5300; 31st day, 4000 f/mg DL), decreased to 1100 ± 800 f/mg DL at 180 days, and tended to remain constant thereafter (2000 ± 1000 f/mg DL). For MMVF ≥5 µm in length, the average fiber concentrations from day 1 sacrifices ranged from 11,500 f/mg DL to 37,600 f/mg DL. PRs of MMVF >5 µm at day 31 ranged from 42 ± 3% to 87 ± 19% and were about equal by day 180.

Retentions at day 545 approached background levels observed in lung tissues from unexposed animals. Retention of MMVF particles <5 µm in length generally exceeded the corresponding PR's of MMVF ≥5 µm long. Likewise, fibers <0.5 µm in diameter had consistently higher PRs than fibers >0.5 µm in diameter at each corresponding sacrifice point. Day 1 concentrations of fibers >20 µm in length ranged from 1500 ± 1700 f/mg DL (Fiber 10) to 4700 ± 600 f/mg DL (Fiber 11). The PRs for Fiber 21 were higher than the average PR for the other MMVF both at 31 and 90 days; but by day 180 the PR had decreased to values close to those of the other MMVF. At 545 days, the PRs for MMVF >20 µm in length were near background level.

Conclusions:

This study showed that MMVFs are more biosoluble than crocidolite. Also, no mortality in rats was observed after short-term exposure (5 days) to MMVFs up to 545 days after last exposure.

Executive summary:

In a short-term inhalation study, Fischer 344 rats were exposed nose-only to four MMVFs (2 glass wools, 1 rock/stone wool and 1 slag wool; no mention of fibres fulfilling Note Q in this study) as well as crocidolite (positive control) at gravimetric concentrations of 30 mg/m³) for 6 hours/day for 5 days. Rats were sacrificed at 1 hour, and 1, 5, 31, 90, 180, 270, 365, and 545 days after exposure to MMVF and lung tissues were examined to assess biopersistence of the test material. Dissolution in vivo suggested as MMVF fibres ≥0.5 µm in diameter and ≥20 µm in length dissolved within 270 days post-exposure. As these decreased, the percentages observed for fibres <0.5 µm in diameter and <5 µm in length increased, which is evidence for the thicker and longer fibres dissolving or breaking over time. Very few MMVF fibres ≥20 µm in length were detected after 545 days. 26% of lung tissues examined contained any MMVF when examined at 545 days, and was almost all siliceous and with the average concentration of <200 f/mg DL. In comparison to crocidolite, MMVF dissolved or fractured in vivo whereas crocidolite fibers did not change.

Reference 9

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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: 13-week exposure was designed to determine and substantiate the maximum tolerated dose of fibrous glass for rats exposed chronically by inhalation and to establish short-term indicators that can be used to predict the maximum tolerable dose for chronic inhalation studies.
- Short description of test conditions: Fischer 344 rats were dosed (nose-only) 0.003, 0.016, 0.03, 0.045 and 0.06 mg/L of test material for 6 h/day, 5 days/week for 13 weeks
- Parameters analysed / observed: Pulmonary pathology, lung epithelium cell proliferation, lung fiber burden, lung lavage cells and chemistry

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

glass fibre

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 8 weeks
- Weight at study initiation: not specified
- Housing: Rats were housed individually in polycarbonate cages containing hardwood bedding in Hazelton 2000 chambers in rooms operated under negative pressure (-20 mm H2O))
- Diet (e.g. ad libitum): ad libitum, pelleted standard Kliba 343 rat maintenance diet (Klingentalmuche AG, Switzerland)
- Water (e.g. ad libitum): ad libitum, fresh water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each animal is supplied fresh aerosol and the air exhaled by one animal does not reach any other animal in the chamber; More details not described: method according to Sachsse et al. 1976
- System of generating particulates/aerosols: Research and Consulting Company (RCC, Geneva) fiber aerosol generation system
- Temperature, humidity, pressure in air chamber: method according to Sachsse et al. 1976 (in German)
- Air flow rate: The exposure chambers provide a positive pressure laminar flow to each animal individually
- Air change rate: method according to Sachsse et al. 1976 (in German)
- Method of particle size determination: For bivariate length/diameter analysis, a sample of each concentration of fiber glass aerosol was collected onto a Nuclepore filter. The filter was sealed in a glass bottle and stored dry until analysis. At the time of analysis, the filter surface was washed into the bottle to retain all fibers, and then filters were ashed and the ash was added back to the fiber suspension in the bottle. The suspensions were diluted to 250 mL with distilled water and dispersed by sonication. Aliquots were filtered onto 0.2-mm pore size Nuclepore membranes. After drying, the membranes were applied to scanning electron microscopy (SEM) stubs and gold coated. Bivariate length and diameter distributions were measured according to the method outlined in WHO Monograph 4 for measuring airborne man-made mineral fibers (WHO, 1985) using scanning electron microscopy (SEM). Diameters were measured at 5000x and lengths at ≥2000x, in a minimum of 20 fields and a maximum of 100 fields or 200 fiber ends, on either a JEOL T 300 SEM or JEOL 840 SEM equipped with a Videoplan Image Analysis System. Magnifications were reduced during length measurement of very long fibers so that the entire length was measured (i.e., lengths were not truncated). Counts were made of both ‘‘total fibers’’ and ‘‘WHO fibers.’’ Total fibers were defined as all particles having a length/diameter ratio ≥3. WHO fibers (i.e., respirable fibers as defined by WHO, 1985) were those fibers having a length/diameter ratio ≥3, diameter <3 µm, and length >5 µm.

- Treatment of exhaust air: not described

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol samples for the FG-exposed groups and the air-only control group were collected on filters in the same location as the animal exposure port. Sampling was performed isoaxially, directly from the output of one of the laminar flow supply tubes to eliminate isokinetic sampling bias. Fiber mass concentrations (mg fiber/m^3 air) were determined for each FG aerosol concentration once during pretest, and at least four times per week during the 13-week exposure period. For measuring fiber mass, aerosol samples were collected onto Gelman membrane filters. The number of fibers/cc air was determined at least once during pretest and once per week throughout the exposure period as follows: Samples were collected on Millipore filters, clarified by acetone vapor, placed between glass slides, and then counted using a Bausch and Lomb Balpan phase contrast microscope at a magnification of 400x. World Health Organization Monograph 4 counting rules were applied for counting the number of WHO fibers/cc (WHO, 1985) once/week. In addition to WHO fibers, the total number of fibers and nonfibrous particles was also counted on one filter every 2 weeks. Additionally, to ensure the uniformity of exposure during each 6-hr exposure period, the fiber concentrations were also monitored continuously using a RAS (MIE Inc., Bedford, MA) light scattering monitor.

- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: not described
- Composition of vehicle: air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aerosol samples were collected onto Gelman membrane filters and the number of fibers/cc air was determined at least once during pretest and once per week through exposure period. Samples were collected on Millipore filters, clarified by acetone vapor, placed between glass slides, then counted using a phase contrast microscope at a magnification of 400x. World Health Organization Monograph 4 counting rules were applied for counting the number of WHO fibers/cc once/week. In addition to WHO fibers, the total number of fibers and nonfibrous particles was also counted on one filter every 2 weeks. The fiber concentrations were also monitored continuously using a RAS light scattering monitor.

Duration of treatment / exposure:

13 weeks; then 10 weeks without aerosol exposure for recovery

Frequency of treatment:

6 hr/day, 5 days/week

Dose / conc.:

3 mg/m³ air (nominal)

Dose / conc.:

16 mg/m³ air (nominal)

Dose / conc.:

30 mg/m³ air (nominal)

Dose / conc.:

45 mg/m³ air (nominal)

Dose / conc.:

60 mg/m³ air (nominal)

Dose / conc.:

3.2 mg/m³ air (analytical)

Dose / conc.:

16.5 mg/m³ air (analytical)

Dose / conc.:

30.5 mg/m³ air (analytical)

Dose / conc.:

44.5 mg/m³ air (analytical)

Dose / conc.:

62.2 mg/m³ air (analytical)

No. of animals per sex per dose:

31 or 36 male rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: random

Positive control:

none

Observations and examinations performed and frequency:

Not described

Sacrifice and pathology:

Randomly selected animals were terminated at 7 weeks, 13 weeks, 19 weeks, and 23 weeks.
- Lung examined microscopically for pathological changes
- Lung fiber burdens were determined
- Cell proliferation in terminal airways quantified in 10-week recovery animals (23 weeks) using PCNA assay.
- Bronchoalveolar lavage analyses

Other examinations:

Lung clearance of microspheres: After 13 weeks exposure, 6 animals from each group were exposed to radiolabeled (85-Sr) microspheres by intratracheal inhalation. The animals were maintained with no further exposure for 10 weeks and whole-body radioactivity was measured weekly (or more frequently than weekly).

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal clinical signs

Mortality:

not examined

Body weight and weight changes:

not examined

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 specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No lung or pleural fibrosis observed in any exposure group

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic evidence of pulmonary change was seen at first termination time point after 7 weeks: 2/6 rats exposed to 60 mg/m^3 fibrous glass showed few small foci over surface of lung. Lesions were more exposed after 13-week exposure. These lesions were not found after 7 or 10 weeks of recovery.
Fibres and fibre fragments seen in many macrophages and almost all micro-granulomas (>3 mg/m^3); unusual clumps of macrophages in the 45 and 60 mg/m^3 exposed rats; microgranulomas found in 1/5 rats exposed to 3 mg/m^3, 4/6 rats at 16 mg/m^3, all rats in the 30, 45, 60 mg/m^3 groups. Following 13 weeks exposure and 7 weeks recovery (without exposure), the number of alveolar macrophages had decreased in all exposure groups. However, the unusual clumps of macrophages in the 45 and 60 mg/m^3 exposed rats were still evident. The number of microgranulomas had also decreased and were only observed in rats exposed to 30, 45, and 60 mg/m^3. In addition, metaplasia (bronchiolization) was observed in the epithelium lining occasional proximal alveoli in two rats in both the 45 and 60 mg/m^3 exposure groups. In this metaplasia, cells had changed from a normal squamous appearance to cuboidal. Early interstitial fibrosis was observed in one rat in both the 45 and 60 mg/m^3 exposure groups. After 13 weeks exposure followed by 10 weeks recovery, further regression in the number of pulmonary macrophages was noted, although the clumps of macrophages noted at the end of the exposure period (13 weeks) in the 45 and 60 mg/m^3 rats were still present. Microgranulomas were found in four of six rats in each of the three highest exposure groups. A minimal amount of bronchiolization was noted in one rat in each of the dose groups exposed to 16 mg/m^3 or greater. Finally a minimal amount of interstitial fibrosis was found in one rat at 16 mg/m^3, in one rat at 45 mg/m^3, and in two of six rats exposed to 60 mg/m^3.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Metaplasia (bronchiolization) observed in epithelium lining with early interstitial fibrosis observed in one rat in each of the 45 and 60 mg/m^3 exposure groups.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung cell proliferation
Lung cell proliferation was assessed by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). After 13 weeks exposure, the percentage of proliferating cells was significantly elevated (compared to positive controls) in all but the 3 mg/m^3 exposure. PCNA immunoreactive nuclei were most abundant in what appeared to be epithelial cells of the terminal bronchioles and alveolar ducts, but were also noted in alveolar macrophages, endothelial and smooth muscle vascular cells, and interstitial cells. Proliferating cells were most numerous at the bronchoalveolar duct bifurcations where fiber-induced inflammatory changes were most prominent. After the 10-week recovery period, no significantly elevated levels of lung cell proliferation were found in any fiber-exposed groups.

Details on results:

In this study, following changes in lung structure and function occurring at the higher doses could be considered: Histopathological changes in the three highest dose groups attest to chronic inflammation; these changes include an influx of phagocytic cells, microgranulomas, and minimal fibrosis. Abnormal clumps of macrophages in the lungs were observed in the 45 and 60 mg/m³ groups, suggest macrophage clearance impairment at these exposure levels. Lung toxicity was also indicated by increased total protein, LDH, NAG and neutrophils in the BAL at exposures≥16 mg/m³ as well as elevated cell proliferation, presence of cuboidal cells and metaplasia in the lung epithelium in the higher dose groups. A lung defense impairment was demonstrated by a dose-dependent retardation of microsphere clearance during the post exposure recovery phase. These changes in lung structure and functions are strong indicators that the maximum tolerated dose at 30 mg/m³ has been reached. Increasing exposure above the level of 30 mg/m³ could only be expected to lead to further overwhelming of defense mechanisms, loss of pulmonary homeostasis and induction of nonspecific lung pathology.

Dose descriptor:

other: Maximum tolerated dose for further studies

Effect level:

30 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

immunology

Critical effects observed:

not specified

Lung fiber burden

Fibers recovered from the lungs also had no significant concentration- or time-dependent dimensional differences. Lung fibers were, however, shorter and thinner than the aerosol fibers. For example, aerosol fiber arithmetic mean dimensions were 1.0 ± 0.6 µm x 21 ± 20 µm, while the lung fiber arithmetic mean dimensions were 0.6 ± 0.3 µm x 7 ± 5 µm (60 mg/m^3 concentration). The lung fiber distribution is more concentrated in the thin-short range, and the small, out-lying peaks are absent, indicating that the respiratory system has selected only the smaller-sized fibers for entry into the deep lung. In the distribution, there are substantial quantities of very long and/or very thick fibers. The number of fibers in the lung was both dose- and time-dependent. For all doses examined, lung fiber concentration increased during the 13-week exposure and decreased during the 10-week post-exposure period. The number of fibers recovered from the lungs (WHO fibers/lung) in each of the five exposure groups was directly proportional to the number of fibers in the aerosol.

Bronchoalveolar lavage

Bronchoalveolar lavage (BAL) fluid from rats after 13 weeks of aerosol exposure demonstrated dose-dependent increased in LDH and NAG activity and total protein concentration. By the 23rd week (13 weeks aerosol exposure plus 10 weeks of recovery), however, no significant dose-dependent differences were observed in any of these three parameters. Some of the cell types in the BAL demonstrated a similar effect. After 13 weeks exposure, the percentage of neutrophils and lymphocytes increased with fiber aerosol concentration. Neutrophil concentration was more than 10-fold greater in the rats exposed to 60 mg/m^3than in the air control animals: mean numbers in millions per lung were 0.07 ± 0.05 for controls, 0.54 ± 0.27 for the 30 mg/m^3exposure, and 0.78 ± 0.08 for the 60 mg/m^3exposure. Lymphocyte elevations were less striking: mean lymphocyte numbers in millions per lung ranged from 0.08 ± 0.08 in the controls to 0.287 ± 0.06 in the 60 mg/m^3exposure. After 10 weeks recovery, cell levels in all fiber groups were similar to the air control cell levels (data not shown). Macrophages constituted the majority of BAL cells (86–97%), and their absolute concentrations were similar in all dose groups at both time points (at the 13-week time point, average macrophages in millions per lung ranged from 7 ± 2 in controls to 8 ± 3 in the highest exposure; at 23 weeks the figure was 10–11 million per lung).

Conclusions:

In this study, treatment of MMVF10 by inhalation lead to effects in the lung of rats. Based on the findings in this study, a maximum tolerated dose of 30 mg/m³ can be considered for further chronic inhalation studies.

Executive summary:

In a subchronic inhalation study, MMVF10 (fibre) in air was administered to male Fischer 344/N rats (31 or 36 per dose) by nose-only exposure at concentrations of 0.003, 0.016, 0.03, 0.045, and 0.06 mg/L (nominal) for 6 hr/day, 5 days/week for 13 weeks.

The test animals were observed for 10 weeks with no exposure following the 13-week aerosol exposure. In this study, following changes in lung structure and function occurring at the higher doses could be considered: Histopathological changes in the three highest dose groups attest to chronic inflammation; these changes include an influx of phagocytic cells, microgranulomas, and minimal fibrosis. Abnormal clumps of macrophages in the lungs were observed in the 45 and 60 mg/m³ groups, suggest macrophage clearance impairment at these exposure levels. Lung toxicity was also indicated by increased total protein, LDH, NAG and neutrophils in the BAL at exposures ≥16 mg/m³ as well as elevated cell proliferation, presence of cuboidal cells and metaplasia in the lung epithelium in the higher dose groups. A lung defense impairment was demonstrated by a dose-dependent retardation of microsphere clearance during the post exposure recovery phase. These changes in lung structure and functions are strong indicators that the maximum tolerated dose at 30 mg/m³ has been reached. Increasing exposure above the level of 30 mg/m³ could only be expected to lead to further overwhelming of defense mechanisms in rat, loss of pulmonary homeostasis and induction of nonspecific lung pathology.

Reference 10

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

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/m³) 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 repeated dose toxicity data of MMVF 10 for Note Q MMVFs using a read-across approach.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal clinical signs

Mortality:

not examined

Body weight and weight changes:

not examined

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 specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No lung or pleural fibrosis observed in any exposure group

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic evidence of pulmonary change was seen at first termination time point after 7 weeks: 2/6 rats exposed to 60 mg/m^3 fibrous glass showed few small foci over surface of lung. Lesions were more exposed after 13-week exposure. These lesions were not found after 7 or 10 weeks of recovery.
Fibres and fibre fragments seen in many macrophages and almost all micro-granulomas (>3 mg/m^3); unusual clumps of macrophages in the 45 and 60 mg/m^3 exposed rats; microgranulomas found in 1/5 rats exposed to 3 mg/m^3, 4/6 rats at 16 mg/m^3, all rats in the 30, 45, 60 mg/m^3 groups. Following 13 weeks exposure and 7 weeks recovery (without exposure), the number of alveolar macrophages had decreased in all exposure groups. However, the unusual clumps of macrophages in the 45 and 60 mg/m^3 exposed rats were still evident. The number of microgranulomas had also decreased and were only observed in rats exposed to 30, 45, and 60 mg/m^3. In addition, metaplasia (bronchiolization) was observed in the epithelium lining occasional proximal alveoli in two rats in both the 45 and 60 mg/m^3 exposure groups. In this metaplasia, cells had changed from a normal squamous appearance to cuboidal. Early interstitial fibrosis was observed in one rat in both the 45 and 60 mg/m^3 exposure groups. After 13 weeks exposure followed by 10 weeks recovery, further regression in the number of pulmonary macrophages was noted, although the clumps of macrophages noted at the end of the exposure period (13 weeks) in the 45 and 60 mg/m^3 rats were still present. Microgranulomas were found in four of six rats in each of the three highest exposure groups. A minimal amount of bronchiolization was noted in one rat in each of the dose groups exposed to 16 mg/m^3 or greater. Finally a minimal amount of interstitial fibrosis was found in one rat at 16 mg/m^3, in one rat at 45 mg/m^3, and in two of six rats exposed to 60 mg/m^3.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Metaplasia (bronchiolization) observed in epithelium lining with early interstitial fibrosis observed in one rat in each of the 45 and 60 mg/m^3 exposure groups.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung cell proliferation
Lung cell proliferation was assessed by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). After 13 weeks exposure, the percentage of proliferating cells was significantly elevated (compared to positive controls) in all but the 3 mg/m^3 exposure. PCNA immunoreactive nuclei were most abundant in what appeared to be epithelial cells of the terminal bronchioles and alveolar ducts, but were also noted in alveolar macrophages, endothelial and smooth muscle vascular cells, and interstitial cells. Proliferating cells were most numerous at the bronchoalveolar duct bifurcations where fiber-induced inflammatory changes were most prominent. After the 10-week recovery period, no significantly elevated levels of lung cell proliferation were found in any fiber-exposed groups.

Details on results:

In this study, following changes in lung structure and function occurring at the higher doses could be considered: Histopathological changes in the three highest dose groups attest to chronic inflammation; these changes include an influx of phagocytic cells, microgranulomas, and minimal fibrosis. Abnormal clumps of macrophages in the lungs were observed in the 45 and 60 mg/m³ groups, suggest macrophage clearance impairment at these exposure levels. Lung toxicity was also indicated by increased total protein, LDH, NAG and neutrophils in the BAL at exposures≥16 mg/m³ as well as elevated cell proliferation, presence of cuboidal cells and metaplasia in the lung epithelium in the higher dose groups. A lung defense impairment was demonstrated by a dose-dependent retardation of microsphere clearance during the post exposure recovery phase. These changes in lung structure and functions are strong indicators that the maximum tolerated dose at 30 mg/m³ has been reached. Increasing exposure above the level of 30 mg/m³ could only be expected to lead to further overwhelming of defense mechanisms, loss of pulmonary homeostasis and induction of nonspecific lung pathology.

Dose descriptor:

other: Maximum tolerated dose for further studies

Effect level:

30 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

immunology

Critical effects observed:

not specified

Lung fiber burden

Fibers recovered from the lungs also had no significant concentration- or time-dependent dimensional differences. Lung fibers were, however, shorter and thinner than the aerosol fibers. For example, aerosol fiber arithmetic mean dimensions were 1.0 ± 0.6 µm x 21 ± 20 µm, while the lung fiber arithmetic mean dimensions were 0.6 ± 0.3 µm x 7 ± 5 µm (60 mg/m^3 concentration). The lung fiber distribution is more concentrated in the thin-short range, and the small, out-lying peaks are absent, indicating that the respiratory system has selected only the smaller-sized fibers for entry into the deep lung. In the distribution, there are substantial quantities of very long and/or very thick fibers. The number of fibers in the lung was both dose- and time-dependent. For all doses examined, lung fiber concentration increased during the 13-week exposure and decreased during the 10-week post-exposure period. The number of fibers recovered from the lungs (WHO fibers/lung) in each of the five exposure groups was directly proportional to the number of fibers in the aerosol.

Bronchoalveolar lavage

Bronchoalveolar lavage (BAL) fluid from rats after 13 weeks of aerosol exposure demonstrated dose-dependent increased in LDH and NAG activity and total protein concentration. By the 23rd week (13 weeks aerosol exposure plus 10 weeks of recovery), however, no significant dose-dependent differences were observed in any of these three parameters. Some of the cell types in the BAL demonstrated a similar effect. After 13 weeks exposure, the percentage of neutrophils and lymphocytes increased with fiber aerosol concentration. Neutrophil concentration was more than 10-fold greater in the rats exposed to 60 mg/m^3than in the air control animals: mean numbers in millions per lung were 0.07 ± 0.05 for controls, 0.54 ± 0.27 for the 30 mg/m^3exposure, and 0.78 ± 0.08 for the 60 mg/m^3exposure. Lymphocyte elevations were less striking: mean lymphocyte numbers in millions per lung ranged from 0.08 ± 0.08 in the controls to 0.287 ± 0.06 in the 60 mg/m^3exposure. After 10 weeks recovery, cell levels in all fiber groups were similar to the air control cell levels (data not shown). Macrophages constituted the majority of BAL cells (86–97%), and their absolute concentrations were similar in all dose groups at both time points (at the 13-week time point, average macrophages in millions per lung ranged from 7 ± 2 in controls to 8 ± 3 in the highest exposure; at 23 weeks the figure was 10–11 million per lung).

Conclusions:

In this study, treatment of MMVF10 by inhalation lead to effects in the lung of rats. Based on the findings in this study, a maximum tolerated dose of 30 mg/m³ can be considered for further chronic inhalation studies.

Executive summary:

In a subchronic inhalation study, MMVF10 (fibre) in air was administered to male Fischer 344/N rats (31 or 36 per dose) by nose-only exposure at concentrations of 0.003, 0.016, 0.03, 0.045, and 0.06 mg/L (nominal) for 6 hr/day, 5 days/week for 13 weeks.

The test animals were observed for 10 weeks with no exposure following the 13-week aerosol exposure. In this study, following changes in lung structure and function occurring at the higher doses could be considered: Histopathological changes in the three highest dose groups attest to chronic inflammation; these changes include an influx of phagocytic cells, microgranulomas, and minimal fibrosis. Abnormal clumps of macrophages in the lungs were observed in the 45 and 60 mg/m³ groups, suggest macrophage clearance impairment at these exposure levels. Lung toxicity was also indicated by increased total protein, LDH, NAG and neutrophils in the BAL at exposures ≥16 mg/m³ as well as elevated cell proliferation, presence of cuboidal cells and metaplasia in the lung epithelium in the higher dose groups. A lung defense impairment was demonstrated by a dose-dependent retardation of microsphere clearance during the post exposure recovery phase. These changes in lung structure and functions are strong indicators that the maximum tolerated dose at 30 mg/m³ has been reached. Increasing exposure above the level of 30 mg/m³ could only be expected to lead to further overwhelming of defense mechanisms, loss of pulmonary homeostasis and induction of nonspecific lung pathology.

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

Dose descriptor:

NOAEC

30 mg/m³

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study. No deviations.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study included one exposure level of the substance at 30 mg/m3 and a negative control group exposed to filtered air. The exposure duration was 6 hours/day, 5 days/week for 2 years with a subsequent post exposure period lasting approximately until 20% suvival in the filtered air control group.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratory, Raleigh, NC
- Age at study initiation: 7-8 weeks
- Weight at study initiation: approx. 180g
- Fasting period before study: no data
- Housing: individually or in groups of two
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

filtered air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Flow-past exposure chamber. Animals were confined separately in restraint tubes positioned radially at several levels of a vetical aerosol supplytube.
- Method of conditioning air: electron charge neutralization with a Ni-63 line source.
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber and oxygen concetrations were all monitored.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric concentration (mg/m3), WHO Fibers (fibers/cm3) and Fibers L>20µm (fibers/cm3) were monitored throughout the entire exposure period of two years.

Duration of treatment / exposure:

6 hours/day, 5 days/week for 2 years with a subsequent post exposure period lasting approximately until 20% survival in the filtered air control group.

Frequency of treatment:

6 hours/day, 5 days/week for 2 years

Remarks:

Doses / Concentrations:
30 mg/m3
Basis:
other: gravimetric concentration

No. of animals per sex per dose:

140

Details on study design:

- Dose selection rationale: The gravimetric concentration of 30 mg/m3 was selected to obtain a fiber concentration of at least 259 WHO fibers/cm3 throughout the exposure period and for being comparable to the other studies.

Positive control:

No positive control group entered in the study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes: clinical signs, morbidity and mortality
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week during the first 13 wk, then every 2 wk.


NECROPSY was performed on all animals

Sacrifice and pathology:

Scheduled sacrifices after 3, 6, 12, 18 and 24 months of 5 animals per time-point.

Statistics:

Fischer's exact test, one sided.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

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

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEC

Remarks:

(carcinogenicity)

Effect level:

ca. 30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No statistically significant findings of broncho-alveolar hyperplasia, adenomas or carcinomas.

Critical effects observed:

not specified

Summary incidence of broncho-alveolar hyperplasia, bronco-alveolar adenomas, and bronco-alveolar carcinomas in rats at risk for tumour formation:

Group	n	Broncho-alveolar hyperplasia		Adenoma		Carcinoma		Carcinoma + adenoma
		n	%	n	%	n	%	n	%
MMVF note Q fibres	107	6	5.6	5	4.7	0	0.0	5	4.7
Control	107	4	3.7	3	2.8	1	0.9	4	3.7

Note. n, Number of animals "at risk" (defined as the number of animals sacrified at the end of the 12-month exposure period, and the number of animals subsequently found dead or sacrified until the termination of the study, provided that they were exposed for at least 12 months and that their lungs were examined histologically).

Statistical method: Exposed group compared to concurrent control using Fischer's exact test, one-sided. [No statistically significant findings at 5% level.]

Conclusions:

The substance showed minimal collagen deposition in the lungs similar to what could be expected for any biologically inert dust at the same exposure level. It is concluded that the substance does not show any carcinogenic potential in the lungs or pleura.

Executive summary:

This is a comparable to guideline study with no deviations. Furthermore, it was published in a peer-reviewed scientific journal and details on the test materials and experimental design are very well documented. The objective of the study was to assess potential pathogenic and/or oncogenic effects of chronic inhalation exposure to stone wool fibers in rats.

107 male Ficher 344 rats were exposed 6h/day, 5 days/week for 2 years to the substance at 30 mg/m³ by nose-only inhalation of a well-characterized fiber test atmosphere. The study showed that the substance holds neither carcinogenic potential nor any fibrogenic potential in the rat.

Reference 2

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study. No deviations.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Methods of Sachsse et al., (1976); Hesterberg et al., (1991); Bernstein et al., (1993) and Cannon et al., (1983) were followed.

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC.
- Age at study initiation: 6 weeks
- Housing:Polycarbonate cages
- Diet (e.g. ad libitum): Pelleted standard Kliba 343 rat maintenance diet
- Water (e.g. ad libitum): Filtered fresh water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-3 oC
- Humidity (%): 30-70%
- Air changes (per hr): 20 air changes/hr
- Photoperiod (hrs dark / hrs light): 12-hr light/dark

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

Filtered air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Source and rate of air:
- System of generating particulates/aerosols:Research and Consulting Company, Geneva, fiber aerosol generation system.
- Temperature, humidity, pressure in air chamber: 22+-3 oC, 30-70%, -20mm H2O
- Air change rate: 20 air change/hr
- Method of particle size determination: WHO Monograph 4 (WHO, 1985)

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes

During preexposure trials and once every three months thereafter, MMVF note Q fibers at each exposure concentration were captured on filters for determination of fiber length and diameter.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During preexposure trials and once every three months thereafter, MMVF note Q fibres' aerosols at each exposure concentration were captured on filters for determination of fiber length and diameter.

Duration of treatment / exposure:

24 months

Frequency of treatment:

6 hr/day, 5 days/week

Remarks:

Doses / Concentrations:
3, 16, and 30 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

140 (only male rats were used)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: To allow comparison with concurrent studies
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: 6 months
- Section schedule rationale (if not random): Random

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week during the first 13 weeks and at least once a month thereafter.


BODY WEIGHT: Yes
- Time schedule for examinations: Once a week during the first 13 weeks and at least once a month thereafter.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data


CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

Sceduled sacrifices were performed: 3 animals after 3 and 6 moths exposure and 6 animals after 12, 18 and 24 moths exposure.

GROSS PATHOLOGY: No

HISTOPATHOLOGY: Yes
Wagner Pathology Grading Scale was used:
Cellular Change
Normal 1 No lesion
Minimal 2 Macrophage response
Mild 3 Bronchioloization, inflammation, fibrosis
Minimal 4 Minimal
Mild 5 Linking of fibrosis
Moderate 6 Consolidation
Severe 7 Marked fibrosis and consolidation
8 Complete obstruction of most airways

Other examinations:

No data

Statistics:

Pairwise comparison 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. All tests of significance were two-tailed, with no formal adjustment for multiple comparisons. The Student t-test was used for comparisons of the physical characteristics of the fibers.

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 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:

not specified

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

HISTOPATHOLOGY: NON-NEOPLASTIC
There was no evidence of treatment-related interstitial lung fibrosis or pleural fibrosis at any time point in the study. Exposure to the substance was associated with nonspecific inflammatory response (macrophage response) in the lungs that did not appear to progress after 6-12 months of exposure. These cellular changes are reversible and are similar to the effects observed after inhalation of an inert dust. No lung fibrosis was observed.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Exposure resulted in no mesotheliomas and no statistically significant increase in lung tumor incidence when compared to that of negative control group.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant body weight changes or excess mortality during the 2 years of exposure to the substance (data not shown).

Dose descriptor:

NOAEC

Remarks:

lung burden

Effect level:

> 3 - <= 30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Lung burden of the substance after 3-24 months exposure to: 30 mg/m3 (3.72-5.03 fibers/mg dry lung x 10^3), 16 mg/m3 (2.09-3.46 fibers/mg dry lung x 10^3) and 3 mg/m3 (0.35-0.62 fibers/mg dry lung x 10^3), respectively

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Dose descriptor:

NOAEC

Remarks:

(toxicity)

Effect level:

30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEC

Remarks:

(carcinogenicity)

Effect level:

30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No evidence of treatment-related interstitial lung fibrosis or pleural fibrosos. No biologically or statistically significant increase of lung tumor incidence.

Critical effects observed:

not specified

Average Wagner Scores in rats to filtererd air, or MMVF11:

Euthanize/exposure (months)	Air control	MMVF 11
		3 mg/m^3	16 mg/m^3	30 mg/m^3
Continous exposure
3/3	1.0	2.0	2.0	3.0
6/6	1.0	2.0	2.3	3.0
12/12	1.0	2.2	3.0	3.0
18/18	1.0	2.5	3.0	3.0
24/24	1.0	2.5	2.7	2.5
Recovery
24/3	1.0	1.2	2.0	2.0
24/6	1.0	1.5	2.0	2.2
24/12	1.0	2.0	2.0	2.0
24/18	1.0	2.0	2.5	2.2
30/24 c	1.0	2.0	2.2	2.5

c: These animals represent the terminal euthanization which occurred when animal survival was approximately 20%.

Summary of Lung Tumor Findings in Fibrous Glass Study:

Exposure group	At risk b	Adenomas	Carcinomas	Total lung tumors	Mesotheliomas
Controls	123	3 (2.4%)	1 (0.8%)	4 (3.3%)	0
3 mg/m^3	118	3 (2.5%)	1 (0.9%)	4 (3.4%)	0
16 mg/m^3	120	6 (5.0%)	3 (2.5%)	9 (7.5%)	0
30 mg/m^3	112	3 (2.7%)	0	3 (2.7%)	0

b: Only animals that were exposed to fibers for at least 1 year were considered at risk for induction of neoplasms as this was the earliest time point a neoplastic finding was observed in this series of studies.

Conclusions:

Inhalation of MMVF note Q fibres does not have a potential for fibrotic or neoplastic changes in the lungs of rats.

Executive summary:

The study is comparable to a guideline study, and contains all relevant details on the test materials and experimental design. The purpose of the study was to determine the chronic biological effects in Fisher 344 rats of inhaled size-separated respirable fractions of the substance. Rats were exposed either to the substance or to the filtered air (negative control) using nose-only inhalation chambers, 6 hr/day, 5 days/week for 24 months to the concentrations of 3, 16 and 30 mg/m³. Exposure to the substance was associated with a nonspecific inflammatory response in the lungs that did not appear to progress after 6-12 months of exposure. No lung fibrosis was observed in the substance exposed animals, and the exposure resulted in no mesotheliomas and no statistically significant increase in lung tumor incidence.

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

5-day inhalation study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Four different glass wools were evaluated for biopersistence and toxicologic effects following short-term inhalation exposure.
- Short description of test conditions: Rats were exposed nose-only to the test fiber aerosols for 5 days, 6 hours/day and maintained for up to 6 months after exposure.
- Parameters analysed / observed: inflammatory response, lung burden, lung clearance, biopersistence and mortality

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Fiber types used: 901F, 902, JM 901 (MMVF10.1), JM 475 (MMVF33)

The major component of each of the five test fibers is silica.

The glasswools are amorphous (i.e., vitreous). Typical of glasswools, 901F, 902, 901, and 475 are all composed of 57–66% SiO2 and 10–17% Na2O. Compared to the other glasswools, 475 glass is relatively low in CaO, MgO, and Na2O, but relatively higher in ZnO and BaO. Unlike the traditional 901 building insulation, 901F is fluorine free. Compared to the traditional 475, the newer special application fiber, 902, is lower in alumina and higher in the more rapidly dissolving components CaO and Na2O.

JM 901 (MMVF10): 57.40% SiO2, 5.17% Al2O3, 0.072% Fe22O3, 0.03% TiO2, 7.65% CaO, 4.16% MgO, 15.5% Na2O, 1.07% K2O, 0.07% SO3, 8.53% B2O3, 0.68% F2
901F: 57.95% SiO2, 5.00% Al2O3, 0.05% Fe22O3, 0.01% TiO2, 0.02% ZrO2, 0.01% Cr2O3, 6.80% CaO, 4.62% MgO, <0.01% SrO, <0.01% BaO, 16.70% Na2O, 1.08% K2O, 0.06% SO3, 8.48% B2O3
902: 66.44% SiO2, 1.91% Al2O3, 0.054% Fe22O3, 0.041% TiO2, 0.021 ZrO2, 0.002 Cr2O3, 4.63% CaO, 3.18% MgO, 0.003% SrO, 0.17% BaO, 15.95% Na2O, 0.60% K2O, 0.20% SO3, 6.72% B2O3, <0.03% F2
JM475 (MMVF33): 58.63% SiO2, 5.87% Al2O3, 0.043% Fe22O3, 0.012% TiO2, 0.034% ZrO2, 0.004% Cr2O3, 1.74% CaO, 0.24 MgO, 0.11% SrO, 4.98% BaO, 4.02% ZnO, 9.55% Na2O, 3.07% K2O, <0.05% SO3, 11.02% B2O3, 0.62% F2

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: ranged 220-240 g for 901F, 173-220 g for 902 and 901 (biopersistence studies) and 147-188 g for 901F, 475, and positive control amosite (toxicology screening studies)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 0.51 - ca. 0.71 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Fibre aerosol generator and nose-only inhalation exposure system described in separate publication (Bernstein et al., 1995)
- System of generating particulates/aerosols: Fibre aerosol was adjusted to achieve a target concentration of fibres with lengths > 20 µm (F > 20 µm) of at least 100 fibers/cm^3

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test aerosols were monitored for mass, fiber numbers, and bivariate fiber dimensions. Aerosol samples were collected on filters placed in animal exposure ports during testing. Fiber mass concentrations were determined in 2 aerosol samples per day for each of the 5 exposure days. Total fibers were counted in each of 5 daily samples using scanning electron microscopy. Bivariate fiber dimensions measured in 2/5 daily samples according to method outlined by WHO modified for electronic monitoring. Dimensions were measured at on-screen magnification of 7000x in a minimum of 20 fields (approx. 27 µm x 27 µm) for 400 fiber ends (representing measurements of at least 200 fibers). Percentages of fibers in each of several fiber size categories were determined in the 2 daily samples, then used to estimate numbers of fibers in each size category from the total fibers/cc counted in the samples from the remaining 3 days.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days

Dose / conc.:

22 mg/m³ air (analytical)

Remarks:

range 12-32 mg/m³ air
=321-443 WHO fibres/cm³
=0.012-0.032 mg/L

No. of animals per sex per dose:

82-105 male rats exposed to each fibre type, 45-55 rats were exposed to filtered air (the controls)

Control animals:

yes

Details on study design:

Rats were exposed by nose-only inhalation for 5 days/6 hr/day. Post-exposure, rats were maintained for up to 6 months. At 8+ time points during the post-treatment period, 7-15 randomly selected rats/fiber exposure group and 5 control rats were euthanized and evaluated for lung fibre burden (after 1, 2, 3 days; 1 and 2 weeks; 1, 3, 6, months; 45 days (901F only).
Toxicology screening assays were evaluated in 5 rats/group after 1, 10 days, and 1 month of recovery.

Positive control:

Amosite asbestos

Observations and examinations performed and frequency:

Rats were observed daily for mortality and for clinical signs and weighed weekly.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

CLINICAL CHEMISTRY: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes, thoracic histopathology

Other examinations:

Further toxicology screening assays: Pleural-cell proliferation, and pleural and bronchoalveolar lavage (PL and BAL), fluid cytology and clinical chemistry. Each parameter evaluated in 5 rats/exposure group.

Statistics:

Statistical significance of bronchoalveolar and pleural lavage was evaluated by Dunnett test based on pooled variance p < 5%/< 1%.

Clinical signs:

not specified

Mortality:

no mortality observed

Description (incidence):

No mention of fibre-exposed rats that died during or after the 5-day exposure due to severe health effects.

Body weight and weight changes:

not specified

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

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

On recovery day 1, elevations in inflammatory indicators for all fibres were apparent primarily in the bronchoalveolar lavage and only slightly in the pleural lavage. By day 14, elevations in inflammatory indicators were seen in the pleural lavage but not in the bronchoalveolar lavage, for all fibres tested except 901F. On day 29-30, elevations were none/negligible for 901F, 902, and 901, but were striking in bronchoalveolar lavage and pleural lavage for 475 and amosite.
On day 1, all 5 fibres tested induced elevations in several inflammatory parameters, but by one-month post-exposure, symptoms subsided for 901F, 902, and 901, but remained for 475 and amosite.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Exposure-related histopathological effects of glasswools 902 and 901 were only seen on 1 day after the last exposure and limited to fibre-containing microgranulomas in rats exposed to 901 and fibre-containing alveolar macrophages.
After 10 and 30 days, thoracic tissues of exposed rats did not differ from those of the control rats. (901F and 475 not tested in this study). The fibres were associated with initial minimal to slight macrophage aggregation and microgranulomas on recovery day 1.
Wagner scores for 901 and 902 glasswools were normal by 30 days of recovery. Some inflammatory symptoms were associated with 475 and amosite after 30 days; average Wagner scores for animals exposed to 475 and amosite increased slightly.

For individual results see Table 1 in box "Any other information on results incl. tables".

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung burdens: Average lung fibre burdence in the biopersistence studies were 7-12 for WHO fibres and 1.0-1.6 for F >20 µm initial amosite lung burdens tended to be higher than those of the glasswools, especially for total fibres; in millions of total fibres per lung, lung burdens were 10–18 for the glasswools and 23 for amosite.

Details on results:

A lack of lethality or severe health effects after short-term exposure to any of the tested fibres was observed.

Remarks on result:

not determinable because of methodological limitations

Remarks:

Only one concentration tested

Critical effects observed:

not specified

Table 1: Lung histopathology in rats exposed to glasswools or amosite asbestos

Wagner grade: 1 = normal, 2 = macrophage aggregation, 3 = cellularity; 4 = minimal fibrosis, 5 -7 = increasing fibrosis

		Pathology scores
Day after exposure ended	Fibre	Macrophage aggregation	Microgranulomas	Bronchiolization	Wagner grade
Day 1	Air	0	0	0	1
	902	1.2	1.0	0	1
	901	1.0	0	0	1
	901F	1.6	0.8	0	2
Day 14	Air	0	0	0	1
	902	0	0	0	1
	901	0	0	0	1
	901F	0	0.4	0	1.8
	475	0.2	1.8	0	2
	Amosite	1.4	2.0	0	2
Day 29 -30	Air	0	0	0	1
	902	0	0	0	1
	901	0	0	0	1
	901F	0	0	0	1
	475	0.6	1.4	0.4	2.2
	Amosite	0	2.5	1.0	2.6

Conclusions:

No mention of fibre-exposed rats that died during or after the 5-day exposure due to severe health effects. The main effect reported after exposure was increased inflammatory responses in the form of fibre-containing microgranulomas in JM 901-esposed rats and fibre-containing alveolar macrophages in JM 901 or 902-exposed rats seen 1 day after exposure ended. The effects were no longer observed after 10 and 30 days of recovery without fibre exposure (i.e. the effects were reversible).

Executive summary:

In a short-term inhalation study, male Fischer 344 rats (82 to 105 per dose) were exposed to four glass wools (JM 901/MMVF10.1, JM 901F, JM 902, JM 475) and amosite asbestos (positive control) for 5 days, 6 hours/day. The concentration of the fibres ranged between 12 to 32 mg/m³ with the mean concentration of 22 mg/m³. This study demonstrated the lack of lethality or severe health effects after short-term exposure to any of the glass wool fibres. The main effect reported after exposure to the glass wool fibres was increased inflammatory responses in the form of fibre-containing microgranulomas in JM 901-esposed rats and fibre-containing alveolar macrophages in JM 901 or 902-exposed rats seen 1 day after exposure ended. The effects were no longer observed after 10 and 30 days of recovery without fibre exposure (i.e. the effects were reversible).

Reference 4

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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: To assess potential pathogenic and/or oncogenic effects of chronic inhalation exposure to inhaled MMVF34 (HT stone wool) fibres by exposing to rats via inhalation of aerosols and biopersistence of the test material. The publication describes the results of a chronic study (study 1) and an ongoing chronic study (study 2).
- Short description of test conditions: Chronic study 1: Details on the final chronic study are published in McConnell et al. (1994). The main protocol is the same as that described for the ongoing study, except that there are 3 fibre exposure groups (3, 16 and 30 mg/m³) and no specific biopersistence satellite groups were included.
Chronic study 2: Rats are exposed to MMVF34 fibre at 30 mg/m³ for 6 hr/day, 5 days/week for 2 years by nose-only inhalation, with subsequent post-exposure period lasting until approx. 20% survival in the test fibre group.
- Parameters analysed / observed: Adverse effects, necropsy and histopathological findings in relation to fibre exposure and lung-burden data.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Study 1: MMVF34
- stone wool fibre, newly developed commercial insulation wool product
Chemical composition of MMVF34:
- 38.9% SiO2
- 23.2% Al2O3
- 2.1% TiO2
6.7% FeO
15.0% CaO
9.6% MgO
1.9% Na2O
0.8% K2O
0.9% other oxides
in vitro dissolution rates based on Si (ng/cm-2 H-1) of MMVF34: 59 (95% CI 41-77) at pH 7.5; 620 (95% CI 434-806) at pH 4.5

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Albino, SPF Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age at study initiation: 7-8 weeks at delivery
- Weight at study initiation: approx. 180 g at delivery
- Fasting period before study: not specified
- Housing: Animals were housed individual or in pairs in stainless steel wire or polycarbonate cages; optimum hygienic conditions behind a barrier system
- Diet (e.g. ad libitum): pelleted standard rat maintenance diet ad libitum during non-exposure periods
- Water (e.g. ad libitum): tap water, ad libitum

DETAILS OF FOOD AND WATER QUALITY: Water supplied was chlorinated community tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: restraint tubes were positioned radially around the flow-past, nose-only exposure chamber
- Method of holding animals in test chamber: confined separately in restraint tubes
- Source and rate of air: not specified
- Method of conditioning air: not specified
- System of generating particulates/aerosols: Fibre aerosols were produced using the RCC Fibre Aerosol Generation System. This system can produce large concentrations of unbroken fibres with little non-fibrous dust.
- Temperature, humidity, pressure in air chamber: Temperature, relative humidity, and oxygen concentrations were monitored continuously.
- Air flow rate: Airflow rate was monitored daily by constant air pressure for the generation and by flow meter at the chamber extraction.
- Air change rate: not specified
- Method of particle size determination: Bulk fibres were size separated, using a water-based process, to be largely rat respirable and to have a geometric mean diameter of approx. 1 µm and a length of approx. 15 µm.
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: not specified
- Samples taken from breathing zone: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric concentrations and fibre numbers were monitored at regular intervals during the studies.

Duration of treatment / exposure:

2 years

Frequency of treatment:

6 hr/day; 5 days/week

Dose / conc.:

30 mg/m³ air (nominal)

No. of animals per sex per dose:

5 animals per exposure time (3, 6, 12, 18, 24 months)

Control animals:

yes, concurrent vehicle

Positive control:

MMVF21 (less biosoluble MMVF than MMVF34)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: clinical signs, morbidity, mortality
Individually examination was performed each week during the first 13 weeks, then every second weeks

BODY WEIGHT: Yes
- Time schedule for examinations: 1x week during first 13 weeks, then every second week

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Lungs removed, weighed and examined under a dissecting microscope
HISTOPATHOLOGY: Yes
- Performed on all animals from the 3, 6, 12, 18-month sacrifices.
- Lungs stained with Trichrome (Masson-Goldner Method) and with Haematoxylin and Eosin
- Lungs were examined and classified histopathologically and given a Wagner score for inflammatory change and fibrosis. [Wagner score: cellular change: 1 = normal; 2 = minimal; 3 = mild; 4 = minimal; 5 = mild; 6 = moderate; 7-8 = severe.
Fibrosis: 4 = minimal; 5 = mild; 6 = moderate; 7-8 = severe]

Other examinations:

Lung burden analysis:
- At necropsy, lung lobes from each animal were removed (from animals in chronic study with histopathology only accessory and right caudal lobe), weighed and frozen. Post-thaw, lobes were finely minced with scissors, dried to a constant weight and the lungs digested using low-temperature ashing. The qualification and quantification of fibre retention relied on lung-burden analyses using SEM.
- Approx. 200-400 fibres per lung sample was analysed. In each group, fibre count for evaluation of lung burden and bivariate analysis of size for evaluation of size distribution were performed on ashed lungs of the animals at each scheduled sacrifice time-point.

Statistics:

Using lung burden data, kinetics were calculated of the elimination of the fibres from the lung by regression analysis of the logarithm of the number of fibres versus time after termination of exposure, corresponding to single exponential model (Fraunhofer-ITA 1997)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

MMVF34: Clinical signs were recorded in isolated animals, not attributed to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

MMVF34: Body weight gains were comparable between the exposed and the air-control groups, but the mean body weight in the control group sacrificed at 3 months was slightly higher than in the exposed group. Potentially related to some discomfort associated with the initial administration of fibres.

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the 3, 6, 12, and 18 months sacrifices, the mean lung weights in the MMVF34 group showed a statistically significant increase compared to control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MMVF34 showed less evidence of pulmonary fibrosis than animals exposed to MMVF21, which itself demonstrated only low levels of pathogenicity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Throughout the 18-month period, only slight macrophage reaction was seen in lungs of rats exposed to MMVF34 (Wagner grade 2). In some animals, occasional micro-granulomas or small areas of bronchiolisation seen at the bifurcations of the terminal or respiratory bronchioles (Wagner grade 3).

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

MMVF34: No information available on development of tumours

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung burden: For MMVF34 long fibres (L>20 µm), a steady state lung burden has apparently been reached by 3 to 6 months exposure.

Details on results:

The carcinogenicity and toxicity results of the chronic study with the positive control MMVF21 suggest that this fibre does not pose a significant health risk to humans and the current results with MMVF34 indicate that this fibre consequently poses an even smaller risk, if any. For MMVF34 throughout the 18 month period, only slight macrophage reaction was seen in the lungs of rats exposed to MMVF34 (Wagner grade 2) although in some animals occasional microgranulomas or small areas of bronchiolization were seen at the bifurcations of the terminal or respiratory bronchioles (Wagner grade 3).

For individual results on lung burdens and pulmonary changes see Table 1 in box "Any other information on results incl. tables".

Remarks on result:

not determinable because of methodological limitations

Remarks:

One concentration used for testing Note Q MMVF

Critical effects observed:

not specified

Table 1: Lung burdens per mg dry lung and pulmonary changes (mean Wagner scores) after different periods of exposure

	Exposure aerosol		Lung burden (Fibres per mg dry lung x 10³)				Interstitial fibrosis (Mean Wagner Score)
Fibre	mg/m³	L > 20 µm/cm³ WHO/cm³	3 months exposure L > 20 µm WHO	6 months exposure L > 20 µm WHO	12 months exposure L > 20 µm WHO	18 months exposure L > 20 µm WHO	3 months exposure	6 months exposure	12 months exposure	18 months exposure
MMVF 21	16.1	74	8	16	37	58	2.2	2.7	2.7	4.0
		150	38	85	210	233
	30.4	114	18	23	55	62	3.2	3.3	3.3	4.0
		243	83	143	319	283
MMVF34	30.5	86	8	11	10	11	1.6	2.6	2.6	2.8
		288	108	147	152	222

Conclusions:

The carcinogenicity and toxicity results of the chronic study with the "positive control" MMVF21 suggest that this fibre does not pose a significant health risk to humans and the current results with MMVF34 indicate that this fibre consequently poses an even smaller risk, if any.

Executive summary:

In a subchronic inhalation study, the two stone wool fibre types MMVF34 (newly developed commercial insulation wool product) and MMVF21 (traditional stone wool) were administered to male Fischer 344/N rats (5 to 6 animals per exposure time) for 6 hr/day; 5 days/week up to two years. Adverse effects, necropsy and histopathological findings in relation to fibre exposure and lung-burden data were examined. In this study, MMVF21 produced a dose-related non-specific inflammatory response and minimal focal pulmonary fibrosis late in the exposure period but no evidence of carcinogenic activity in either the lungs or the pleura. An observed pulmonary fibrosis, in addition to an increase in lung weights, indicates that the maximum tolerated dose had been achieved in this study. Only preliminary histopathological results up to 18 months exposure are available from the chronic inhalation study with MMVF34 but the histopathological changes are minor compared to MMVF21 and based on the available results it seems unlikely that MMVF34 exposed animals will develop pulmonary tumors in excess of those that develop in the control. When comparing the pathology after 3, 6, 12 and 18 months exposure, MMVF34 showed minor histopathological changes compared to MMVF21. The carcinogenicity and toxicity results of the chronic study with MMVF21 suggest that this fibre does not pose a significant health risk to humans, and the current results with MMVF34 indicate that this fibre consequently should pose an even smaller risk, if any.

Reference 5

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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: Acute inhalation toxicity test using rats
- Short description of test conditions: Rats were exposed nose-only to two stone/rock wool fibres (MMVF34 and MMVF21) in aerosol for 5 days, 6 hr/day.
- Parameters analysed / observed: Biopersistence, morbidity and mortality

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test material used: MMVF34

Chemical composition of MMVF34:
- 38.9% SiO2
- 23.2% Al2O3
- 2.1% TiO2
6.7% FeO
15.0% CaO
9.6% MgO
1.9% Na2O
0.8% K2O
0.9% other oxides
in vitro dissolution rates based on Si (ng/cm-2 H-1) of MMVF34: 59 (95% CI 41-77) at pH 7.5; 620 (95% CI 434-806) at pH 4.5

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Albino, SPF Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age at study initiation: 7-8 weeks at delivery
- Weight at study initiation: approx. 180 g at delivery
- Fasting period before study: not specified
- Housing: Animals were housed individual or in pairs in stainless steel wire or polycarbonate cages; optimum hygienic conditions behind a barrier system
- Diet (e.g. ad libitum): pelleted standard rat maintenance diet ad libitum during non-exposure periods
- Water (e.g. ad libitum): tap water, ad libitum

DETAILS OF FOOD AND WATER QUALITY: Water supplied was chlorinated community tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: restraint tubes were positioned radially around the flow-past, nose-only exposure chamber
- Method of holding animals in test chamber: confined separately in restraint tubes
- Source and rate of air: not specified
- Method of conditioning air: not specified
- System of generating particulates/aerosols: Fibre aerosols were produced using the RCC Fibre Aerosol Generation System. This system can produce large concentrations of unbroken fibres with little non-fibrous dust.
- Temperature, humidity, pressure in air chamber: Temperature, relative humidity, and oxygen concentrations were monitored continuously.
- Air flow rate: Airflow rate was monitored daily by constant air pressure for the generation and by flow meter at the chamber extraction.
- Air change rate: not specified
- Method of particle size determination: Bulk fibres were size separated, using a water-based process, to be largely rat respirable and to have a geometric mean diameter of approx. 1 µm and a length of approx. 15 µm.
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: not specified
- Samples taken from breathing zone: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric concentrations and fibre numbers were monitored at regular intervals during the studies.

Replicate fibre samples were subjected to a continuous constant flow (Flow-rate/Initial Surface Area = 0.03 µm/s) of buffer solution (modified Gamble's solution) at two different pH levels in a dissolution rate measurement apparatus. Dissolution rates were calculated on the basis of the analysis of dissolved Si.
The gravimetric concentration was adjusted to obtain a concentration of long fibres (L >20 µm) of 150 fibres/cm³ in order to increase lung burdens and to increase statistical confidence in the results.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hr/day

Dose / conc.:

60 mg/m³ air (analytical)

No. of animals per sex per dose:

66 males

Control animals:

yes, concurrent vehicle

Positive control:

MMVF21 (traditional stone wool fiber)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: clinical signs, morbidity, mortality
Individually examination was performed each week during the first 13 weeks, then every second weeks

BODY WEIGHT: Yes
- Time schedule for examinations: 1x week during first 13 weeks, then every second week

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No

Other examinations:

Lung burden analysis:
- At necropsy, lung lobes from each animal were removed (from animals in chronic study with histopathology only accessory and right caudal lobe), weighed and frozen. Post-thaw, lobes were finely minced with scissors, dried to a constant weight and the lungs digested using low-temperature ashing. The qualification and quantification of fibre retention relied on lung-burden analyses using SEM.
- Approx. 200-400 fibres per lung sample was analysed. In each group, fibre count for evaluation of lung burden and bivariate analysis of size for evaluation of size distribution were performed on ashed lungs of the animals at each scheduled sacrifice time-point.

Statistics:

Using lung burden data, kinetics were calculated of the elimination of the fibres from the lung by regression analysis of the logarithm of the number of fibres versus time after termination of exposure, corresponding to single exponential model (Fraunhofer-ITA 1997)

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse symptoms observed

Mortality:

no mortality observed

Description (incidence):

No adverse symptoms observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse symptoms were 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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Lung burden: The biopersistence results after short-term inhalation exposure showed a rapid removal of MMVF34 long fibres (L > 20 mm) from the lungs compared to MMVF21.

Remarks on result:

not determinable because of methodological limitations

Remarks:

One concentration tested

Critical effects observed:

not specified

Conclusions:

In this study, no effects on morbidity and mortality was observed in rats after 5 days exposure. The biopersistence results after short-term inhalation exposure showed a rapid removal of MMVF34 long fibres (L > 20 mm) from the lungs compared to MMVF21.

Executive summary:

In a subacute inhalation study, the Note Q stone wool fibre type MMVF34 (newly developed commercial insulation wool product) was administered to male Fischer 344/N rats (5 to 6 animals per exposure time) at a dose of 60 mg/m³ in air for 6 hr/day; 5 days/week with subsequent post-exposure periods lasting up to 12 months. Morbidity, mortality and lung burden was examined.

Adverse effects, necropsy and histopathological findings in relation to fibre exposure and lung-burden data were examined. The traditional non-Note Q fibre MMVF21 served as a positive control.

 

No effects on morbidity and mortality was observed in rats after 5 days exposure. The biopersistence results after short-term inhalation exposure showed a rapid removal of MMVF34 long fibres (L > 20 mm) from the lungs compared to MMVF21.

Reference 6

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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: Subchronic inhalation study in rats to evaluate the effects of new biosoluble high-aluminium low silica HT type stone wool. The study was conducted according to the EU protoxol for conducting subchronic inhalation studies in Man-made vitreous fibres (European Joint Research Centre, 1999)
- Short description of test conditions: Male Wistar rats exposed nose-only to 15 mg/m³ aerosol stone/rock wool (MMVF21) and 3 stone wool fibres (RIF41001, RIF42020-6; length >20 µm) for 6 hr/day, 5 days/week for 3 months
- Parameters analysed / observed: Pulmonary change and fibre numbers in lung, including bronchoalveolar lavage fluid for evaluation of inflammatory response and measurement of cell proliferation, assessment of early fibrosis through histological examination and comparison of body weight and lung lobe weights.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

HT wool (high-aluminium low-silica): CAS-No. 287922-11-6
RIF41001: 42.7% SiO2, 18.9% Al2O3, 1.6% TiO2, 6.3% FeO, 18.3% CaO, 7.8% MgO, 1.7% Na2O, 0.9% K2O, 0.2% other oxides
RIF42020-6: 36.8% SiO2, 20.7% Al2O3, 1.4% TiO, 5.4% FeO, 18.0% Cao, 10.2% MgO, 4.4% Na2O, 0.5% K2O, 0.9% other oxides

According to the study by Guldberg et al. (2000), the composition of the stone wool fulfill the criteria for exoneration from classification as a possible carcinogen according to the European Commission Directive from 1997 and/or the German regulation (TRGS 905, 1999) based on the short-term in vivo biopersistence tests.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats recommended by EPA workshop (Vu et al. 1996) for use in subchronic and chronic inhalation toxicity studies of fibres. According to EU guidelines, the use of male Wistar rats is preferred.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 200-300 g
- Housing: Animals were housed in groups of two in polycarbonate cages when not being exposed; optimum hygienic conditions behind barrier system
- Diet (e.g. ad libitum): Pelleted standard rat maintenance diet; ad libitum
- Water (e.g. ad libitum): Tap water; ad libitum
- Acclimation period: 2 weeks, and accustomed to nose-only tubes for 3 weeks pre-exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 40-70%
- Air changes (per hr): air conditioned; not specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 0.54 - <= 0.62 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only, flow-past technique; fiber aerosol is supplied to each animal individually and exhaled air is immediately exhausted.
- Method of holding animals in test chamber: Battelle type polycarbonate tubes; animals keep noses close to airflow at opening of tube. Exposure of animals was performed in identical exposure chambers of cylindrical shape, each housing up to 48 animals.
- Method of conditioning air: airflow, temperature, humidity monitored continuously and were stored as 20 min mean values.
- System of generating particulates/aerosols: High pressure pneumatic disperser, fed with test substances under computerized control, with feedback to the actual aerosol concentrations measured by an aerosol photometer. Aerosols were neutralized by a 63-Ni source to reduce the charge on the fibres.
- Temperature, humidity, pressure in air chamber: Airflow
- Air flow rate: approx. 1 L/min (laminar)
- Method of particle size determination: Lengths and diameters performed using SEM at magnification of at least 2000. Recording of particles was stopped when a total of 30 particles had been counted.

TEST ATMOSPHERE
- Brief description of analytical method used: not necessary to measure oxygen concentration because environment was laminar
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual aerosol concentrations were measured by an aerosol photometer. The photometer gives a scatter light signal, which is nearly proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the studyby comparison with gravimetric and fibre concentration. The aerosols of the test substances were neutralised by a 63Ni source to reduce the charge on the fibres.

Duration of treatment / exposure:

3 months

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

15 mg/m³ air (nominal)

Remarks:

RIF41001: 15.9 mh/m³
RIF42020-6: 14.6 mg/m³

No. of animals per sex per dose:

32 male rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Animals randomized to treatment groups stratified on body weight. 32 rats (including 2 reserve animals) per exposure group. 10 rats allocated for each post-exposure kill group. 5 animals killed at each time point for histopathology also used for the lung burden determinations.

Positive control:

MMVF21

Observations and examinations performed and frequency:

Rats examined daily for clinical signs, morbidity, and mortality. Individually examined outside the cage once a week. Body weight recorded once per week during the first 3 months, then every second week. Lavagate was used to determine biochemical parameters (LDH, beta-glucuronidase and total protein).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

At the scheduled kills the lungs were removed in toto, weighed without the trachea and carefully examined. The right lobes were removed, weighed and deep frozen for lung fibre burden analysis (see below). The left lung lobe was perfused with fixative via the trachea at a pressure of 20 cm water for 2 h.

Other examinations:

Assessments: bronchoalveolar lavage fluid (BALF) for evaluation of inflammatory response (e.g. protein content, enzymes, increase in polymorphnuclear leucocytes) and measurement of cell proliferation, assessment of early fibrosis through histological examination and comparison of body weight and lung lobe weights.

Statistics:

Individual toxicological data were compared to clean air control group. For numeric parameters, the statistics were done using Dunnett's test. For comparison of pathological findings, a pairwise Fischer's test between control and treatment groups was performed.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs recorded in isolated animals, not attributed to treatment with test material.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality was recorded in isolated animals, not attributed to treatment with test material.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gains comparable between exposed and air control group.

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:

effects observed, treatment-related

Description (incidence and severity):

LDH and total protein significantly elevated in all groups at end of exposure kill and remained elevated 3-months post-exposure.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 3 months exposure, mean lung weights in HT and MMVF21 exposed groups were statistically significantly increased compared with control. At the 3-month post-exposure sacrifice, the HT group no longer had significant increase in lung weight, but the MMVF21 did.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Lung fibre burden:
In both stock fibres and aerosol fibres the diameter of the HT fibres was smaller than that of the MMVF21 fibres. However, when the lung fibre burden was examined at the first-time point, the dimension of the retained fibres was very similar for all fibre types. This probably indicates that the lung filters out the thicker fibres, so that the lung tissue is exposed to very similar fibre dimensions regardless of the fibre type. The maximum lung burden achieved was considerably lower in the HT groups than in the MMVF21 group, except in the RIF41001 group, which showed comparable numbers at the end of exposure. Even with this HT type the long fibres cleared much faster in the recovery period than MMVF21.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

All control animals graded 1 (according to Wagner scale). Rats exposed to HT fiber types had lower Wagner scores than animals exposed to MMVF21. Only slight macrophage reaction seen in lungs of rats exposed to HT fibre types (Wagner 1-2). At 3-months' exposure, Wagner grade 1 noted in all animals exposed to RIF41001. In rats exposed to RIF42020-6, Wagner grade 1 was noted in 2 rats and grade 2 noted in 3 rats. Wagner grade 2 characterized by focal minimal microgranulomas at the bronchiolar-alveolar junction. The MMVF21 group had 1 rat with grade 3, 4 rats with grade 4, measured at 3 months' exposure. After 3 months' exposure, differences between HT and MMVF21 were seen for all the histological parameters examined (alveolar bronchiolization, microgranulomas, collagen deposition at bronchiolar-alveolar junction, pleural collagen deposition, macrophages in alveolar lumina). In control group, there were 0 lesions.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Cell proliferation enhanced at end of exposure for MMVF21 for all three labelling indices, but only for the bronchiolar epithelium in RIF41001 group. No significant increase in cell proliferation found at kill dates after the 3-month exposure ended.

Details on results:

For HT and MMVF21, a significant increase in PMN and reduction in percentage of macrophages in bronchoalveolar lavage fluid was observed.
No clear difference in bronchoalveolar lavage fluid cell concentration and percentage of cells between MMVF21 and HT groups, though the percentage of PMNs in the HT groups tended to be higher.

Remarks on result:

not determinable because of methodological limitations

Remarks:

Only one concentration tested

Critical effects observed:

not specified

Conclusions:

In this study, mild pathological changes were found in the lungs of rats exposed with HT. Further changes included alveolar macrophage aggregation and/or microgranulomas at the bronchiol alveolar junction in the few rats affected. No fibrogenic potential was noted of the two HT fibres.

Executive summary:

In a subchronic inhalation study, male Wistar rats (32 per dose) were exposed nose-only to new biosoluble high-aluminum low-silica HT type stone wool (RIF41001 and RIF42020 -6) in air for 6 hours/day, 5 days/week for 3 months, and followed post-exposure for several months. The parameters measured included pulmonary change and fibre numbers in lung, including bronchoalveolar lavage fluid for evaluation of inflammatory response and measurement of cell proliferation, assessment of early fibrosis through histological examination and comparison of body weight and lung lobe weights. After exposure of rats to the new biosoluble fibers (HT), no biologically significant effects were observed except that a statistically significant increase in lung weight was observed up to 1.5 months post-exposure in all 3 treatment groups. At 3 months post-exposure, the small increase was no longer significant. Minimal morphological changes were diagnosed in the HT fibre groups at 3 months and 1.5 and 3 months post-exposure. No fibrogenic potential noted of the two HT fibres. No clear-cut difference between the different biosoluble fibre types noted. Only slight macrophage reaction seen in lungs of rats exposed to HT fibre types: Wagner grade 1 or 2 in all animals examined.

Reference 7

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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: Chronic nose-only inhalation study in Fischer 344/N rats to investigate the potential pathogenic effects of two different types of man-made vitreous fibres.
- Short description of test conditions: Male rats were exposed nose-only to rock wool and slag wool in air for 6 hours/day, 5 days/week for 24 months. Rats were held post-exposure for lifetime observation (until approx. 20% survived).
- Parameters analysed / observed: Clinical signs, morbidity, mortality, necropsy, lung fiber burden measurements, progression of pulmonary lesions during exposure period and post-exposure period.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Slag wool:
- Source: USG Interiors (USA) slag wool
Slag wool chemical composition by weight percent: 38.4% SiO2, 10.6% Al2O3, 38.0% CaO, 9.9% MgO, 0.3% Fe2O3, 0.4% Na2O, 0.5% K2O, 0.5% TiO2, 0.7% MnO, 1.8% S

Other details: Bulk fibers with chemical compositions typical of commercial insulation products were size separated to be largely rat respirable (<1.5 µm diameter and <80 µm length), and to have average dimensions (average aerosol geometric mean diameter <1.0 µm and length >15 µm). Approx. 4000 kg of bulk product was size separated using a water-based process to obtain 10 kg of stock fiber with the required dimensions to be used in the inhalation study.

Species:

rat

Strain:

Fischer 344

Remarks:

344/N

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: 6 weeks old
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: Animals were housed individually in polycarbonate cages containing hardwood bedding in Hazelton 2000 chambers under negative pressure (-20 mm H20)
- Diet (e.g. ad libitum): pelleted feed (Kliba 343, Klingentalmuehle AG, Switzerland); ad libitum
- Water (e.g. ad libitum): filtered fresh water; ad libitum
- Acclimation period: 2 week quarantine period

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

not specified

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Rats placed in exposure tubes
- Method of holding animals in test chamber: confined separtaely in tubes positoined radially around the exposure chamber
- Source and rate of air: Flow-past nose-only sytem 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.
- Method of conditioning air: not specified
- System of generating particulates/aerosols: A system of nondestructive aerosolization to to maximize the number of "rat-respirable fibers". It produces large numbers of unbroken fibers with lower levels of nonfibrous dust.
- Temperature, humidity, pressure in air chamber: levels not specified, but monitored daily
- Method of particle size determination: Fiber mass concentrations were measured at least 4x/week during the 2-year exposure period. Each aerosol sample from each fiber type and dose level was collected. The aerosols were analyzed for WHO fibers per cubic centimeter

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Fiber mass concentrations were measured at least 4 times/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 Gellman membrane filter from one of the laminar flow ports int eh exposure chamber to elimniate isokinetic sampling bias. Aerosol concentrations were monitored continuously during the exposure period using an RAS light-scattering monitor to assure uniformity. The aerosols were analyzed for WHO fibers per cubic centimeter once each exposure week during week 1-13 and every other week thereafter through exposure week 104. The total numbers of fibers and non-fibrous particles were counted once aech month on a filter for each fiber type and dose. Samples were collected on Millipore filters, placed between glass slides, clarified, counted by phase-contrast microscopy at 400x magnification. At least 20 fields counted on each slide. Once per month, total fibers and particles were counted to determine total fibers per cubic centimeter and particles per cubic centimeter.
Every 3 months, samples of slag wool aerosols for each dose were collected on filters for determination of fiber length and diameter.

Duration of treatment / exposure:

24 months

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

3 mg/m³ air (nominal)

Remarks:

gravimetric concentration 3.06 ± 0.44 mg/m³

Dose / conc.:

16 mg/m³ air (nominal)

Remarks:

gravimetric concentration 16.11 ± 1.39 mg/m³

Dose / conc.:

30 mg/m³ air (nominal)

Remarks:

gravimetric concentration 29.89 ± 2.87 mg/m³

No. of animals per sex per dose:

140 rats per exposure group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose (30 mg/m³) was chosen to be comparable to other studies of MMVFs
There were 140 rats in each rock and slag wool exposure group, positive control, unexposed chamber control groups exposed for 6 hr/day, 5 days/week for up to 24 months.
Groups of 6 randomly selected rats, identified at the start of the study, from each exposure group were killed at 3, 6, 12, 18, 24 months for lung fibre burden measurements and to follow progression of pulmonary lesions. In addition, groups of 3-6 rats from each group were removed from exposure at 3, 6, 12, 18 months and held until 24 months, at which time they were sacrificed to ascertain whether there was progression or regression of lesions and to determine lung fibre burdens following cessation of exposure.

Positive control:

Crocidolite asbestos (10 mg/m³)

Observations and examinations performed and frequency:

Test animals were observed daily for cilnical 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 each month after.

Sacrifice and pathology:

At necropsy, the accessory lobe of the lung was removed, weighed, frozen, then thawed, dehydrated, and evaporated to constant weight. The fibers were counted and measured similar to the way the aerosol fiber samples were counted. Other tissues were processed routinely including nasal cavity, larynx, trachea, bronchi, mediastinal, mesenteric lymph nodes, liver, spleen, kidneys, heart, an dall grossly visible lesions. Lungs were examined and classified histopathologically and given a Wagner score for inflammatory change and fibrosis: 1 = normal, 2 = minimal (macrophage response), 3 = mild (macrophage/bronchiolization), 4-8 denote degrees of fibrosis, 4 = minimal (fibrosis restricted to terminal bronchioles/proximal alveoli), 5 = mild (interlobular linking), 6 = moderate (early consolidation), 7 = severe (marked fibrosis/consolidation, 8 = complete obstruction of airways.

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 months (crocidolite for 10 months) were considered at risk for induction of neoplasms. All tests of signficance were two-tailed, with no formal adjustment for multiple comparisons. Student's t-test was used for comparisons of teh physical characteristics of the fibers.

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal clinical signs were observed.

Mortality:

no mortality observed

Description (incidence):

Survival was comparable to unexposed controls.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain was comparable to unexposed controls.

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

Immunological findings:

not examined

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 any of the slag-wool-exposed rats. No lung weight differences observed at 24 months in any of the groups compared to controls, probably due to high incidence of pulmonary leukemia, which is common in older F344/N rats.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic lesions were observed in the lungs or pleura of rats exposed to slag wool at any point during the study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dose- and time-related increase in pulmonary macrophages, microgranuloma formation, and bronchiolization seen. Fibres and fibre fragments found in many macrophages and microgranulomas. However, there was no evidence of fibrosis at any time. No treatment-related lesions were observed in the pleura.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Slag wool: Occasional bronchoalveolar neoplasms were found: 2/116 low dose, 0/115 mid dose, 3/115 high dose, compared to 2/126 unexposed controls. No treatment-related lesions were observed in upper respiratory track or in any other organ. Many rats, including unexposed controls, showed evidence of mononuclear cell leukemia involvement of lung (diffuse gray discoloration, paleness, and/or multifocal punctuate gray foci) at 24 months accompanied by enlarged spleen and nodular liver.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung burden
The average lung burdens of WHO fibres reached their peak at 1 yr and plateaued during the remainder of the study. The retention of slag wool fibres decreased rapidly after exposure ceased. The decrease was mire dramatic for fibres > 20 µm in length for slag wool, which decreased from 31 000 to 200. The electron microscopic examination used in the counting of the fibres also revealed ultrastructural changes in the MMVF fibres after retention in the lung. After 6 months the slag wool fibres showed segmental dissolution and cross cleavage. In contrast, the crocidolite fibres showed no morphologic change, even after 24 months in the lung.

Dose descriptor:

NOAEC

Effect level:

30 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Critical effects observed:

not specified

Exposure atmosphere and fibre characterisation

The three airborne gravimetric concentrations (mg/m³ ± SD) of rock wool averaged 3.06 ± 0.41, 16.14 ± 1.17, and 30.39 ± 2.79 and slag wool 3.06 ± 0.44, 16.11 ± 1.39, and 29.89 ± 2.87 during the 2 year exposure. These concentrations corresponded to average WHO fibre numbers (fibres/cm³ ± SD) of 34 ± 13, 150 ± 51, and 243 ± 67 for rock, and 30 ± 15, 131 ± 40, and 213 ± 62 for slag. The average numbers of rock wool fibres > 10 µm in length were 26 ± 9, 122 ± 33, adn 186 ± 51 and those > 20 µm were 13 ± 5, 74 ± 20, and 114 ± 32. Slag wool fibres > 10 µm in length numbered 20 ± 8, 96 ± 28, and 165 ± 51 and those > 20 µm were 10 ± 4, 50 ± 14, and 99 ± 31. The aerosols also contained nonfibrous particulates. The crocidolite asbestos aerosol (positive control) was 10.04 ± 0.90 mg/m³ which corresponded to an average of 1610 ± 989 WHO fibres/cm³. The average number of fibres/cm³ > 10 µm in length was 677 ± 416, while the number of those > 20 µm was 236 ± 145.

Conclusions:

Non-specific inflammatory response, no evidence of carcinogenic activity in either the lung or pleura. No treatment-related macroscopic lesions were observed in the lungs or pleura of rats to slag wool at any point of the study. Microscopically, there was a dose- and time-related increase in pulmonary macrophages, microgranuloma formation and bronchiolisation. No evidence of fibrosis at any point. No treatment-related lesions were observed in pleura. No biologically significant adverse health effects observed for the test material MMVF22.

Executive summary:

In a chronic inhalation study, male Fischer 344/N rats (160 per dose) were exposed nose-only to slag wool MMVF22 (a Note Q MMVF) in air for 6 hours/day, 5 days/week for 24 months. Rats were held post-exposure for lifetime observation (until approx. 20% survived). Crocidolite asbestos (10 mg/m³) was used as a positive control. Clinical signs, morbidity, mortality, necropsy, lung fibre burden measurements and progression of pulmonary lesions during exposure period and post-exposure period were investigated. No abnormal clinical signs, changes in body weight or mortality were observed. Non-specific inflammatory response, no evidence of carcinogenic activity in either the lung or pleura. No treatment-related macroscopic lesions were observed in the lungs or pleura of rats to slag wool at any point of the study. Microscopically, there was a dose- and time-related increase in pulmonary macrophages, microgranuloma formation and bronchiolisation. No evidence of fibrosis at any point. No treatment-related lesions were observed in pleura. No biologically significant adverse health effects observed for the test material MMVF22.

Reference 8

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

5 days exposure

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Short-term inhalation study in rats to determine biopersistence of the test material by analyzing the concentrations in lung tissue
- Short description of test conditions: Rats were exposed nose-only to 30 mg/m³ of two fiber glass compositions (rockwool and slagwool) with filtered air as a control 6 h/day for 5 days. Animals were sacrificed at various time intervals ranging from 1 hour to 545 days post-exposure and fibers were recovered from digested lung tissue to determine changes in concentrations and fiber retention.
- Parameters analysed / observed: Digested lung tissue analyzed to determine changes in concentrations (fibers/mg dry lung) and fiber retentions (expressed as percent of day 1 retention [PR]) for selected dimension categories.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Glasswool:
- Fiber 10, source: Manville Corp.
- Fiber 11, source: Certainteed Corp.

Rockwool:
- Fiber 21, source: Roxul International

Slagwool:
- Fiber 22, source: USG Interiors

From each MMVF bulk product, the fraction selected had fibers of average dimensions of approximately 1 µm diameter and 15 µm length. Fibers of such dimensions are found in workplace aerosols. The fibers obtained accounted for 0.25 to 5 wt-% of the bulk product, depending on the initial diameter distribution of the MMVF.

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

no data

Sex:

not specified

Details on test animals or test system and environmental conditions:

no data

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: filtered air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Fiber aerosols were lofted using a generation system designed not to break, grind, or contaminate the fibers
- Method of particle size determination: optical and scanning electron microscopy was used for length and diameter dimensions. Diameters were measured at x5000 magnification, from a
minimum of 200 fields or 100 counted fibers. Length measurements were made by optical microscopy at x1500 magnification to reduce the risk of truncated fibers.

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol concentrations for fiber mass were monitored by gravimetric sampling.

- Samples taken from breathing zone: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Fiber concentration levels (fibers/cm^3) were determined by phase contrast microscopy (x400 magnification) for total and WHO fiber numbers.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hr/day

Dose / conc.:

30 mg/m³ air

Remarks:

gravimetric concentration

No. of animals per sex per dose:

9 animals per dose (sex not specified)

Control animals:

yes

Details on study design:

Groups of 9 rats were exposed "nose-only" for 6 hr/day for 5 days to one concentration of 30 mg/m3 for each MMVF. Another group was exposed to 10 mg/m3 of crocidolite fibers and another group breated filtered air.

Positive control:

no data

Observations and examinations performed and frequency:

Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: No

Other examinations:

Tissue concentrations:
9 rats for each fiber type and 9 controls were sacrificed at 1 hour, 1, 5, 31, 90, 180, 270, 365, 545 days after fiber inhalation ended. Lungs were removed, infracardiac lobes separated, and remaining lung tissues preserved. Lobes from 5 rats from each exposed group and from the controls were dehydrated and dried to constant weight to obtain the initial dry weight of tissue for determination of fiber concentrations. Tissues were low temperature ashed, fibers recovered, fiber number, diameters and lengths were determined via SEM and optical microscopy.

Statistics:

Concentrations of fibers per mg of dry lung tissue (f/mg DL) were calculated, as were concentrations of fibers and the percentages of day 1 retention (or PR) for selected diameter and length categories for each sacrifice time point.

Clinical signs:

not examined

Mortality:

no mortality observed

Body weight and weight changes:

not examined

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

See box "Any other information on results incl. tables".

Details on results:

Results from this study indicated that, of the long crocidolite fibers retained in the pulmonary region at day 1, approximately 40% remained through 545 days.
The nearly complete disappearances of MMVF fibers ≥0.5 µm in diameter and >20 µm in length within 270 days postexposure strongly suggested dissolution. These disappearances were accompanied by increases in the percentages observed for fibers <0.5 µm in diameter and <5 µm in length, suggesting that the thicker and longer fibers had dissolved or broken with time, "enriching" the thinner and shorter fractions. In addition, none, or very few MMVF fibers ≥20 µm in length were detected in the lung tissues from exposed animals at 545 days, whereas for crocidolite, fiber concentration at 545 days remained at 2000 ± 400 f/mg DL.

Remarks on result:

not determinable because of methodological limitations

Remarks:

Only one concentration tested

Critical effects observed:

not specified

Geometric mean diameters

The average diameters and lengths of crocidolite fibers recovered from the rat lungs at various retention times differed little, whereas the MMVF showed a decrease in both geometric mean diameter and length with time retained in the animals' lungs.

Fiber concentrations in lung

Fibers recovered from the 1-hr sacrifice represent the deposition in both the conducting airway and alveolar regions of the lung, since particle clearance from the conducting airways is assumed to be complete within 24 hr after stopping exposure. For crocidolite fibers ≥5 µm in length, the average fiber concentrations from the day 1 sacrifice were 168,000 ± 47,000 and for 31, 100,000 ± 31,000 f/mg DL. The average decreased to 58,000 ± 12,000 at day 365 sacrifice and 56,000 ± 6000 at day 545.

Long crocidolite fiber retention tended to be prolonged compared to retention of crocidolite particles (i.e., <5 µm long) as indicated by higher retention values for the longer fibers beginning after 31 days. Percent retentions of fibers≥10 µm did not differ significantly from the PRs of fibers ≥5 µm. PRs of fibers >20 µm in length tended to be higher than those for fibers ≥5 µm at 365 and 545 days. For crocidolite fibers >20 µm in length, average concentrations were roughly equal between days 1 and 31 (first day, 5300; 31st day, 4000 f/mg DL), decreased to 1100 ± 800 f/mg DL at 180 days, and tended to remain constant thereafter (2000 ± 1000 f/mg DL). For MMVF ≥5 µm in length, the average fiber concentrations from day 1 sacrifices ranged from 11,500 f/mg DL to 37,600 f/mg DL. PRs of MMVF >5 µm at day 31 ranged from 42 ± 3% to 87 ± 19% and were about equal by day 180.

Retentions at day 545 approached background levels observed in lung tissues from unexposed animals. Retention of MMVF particles <5 µm in length generally exceeded the corresponding PR's of MMVF ≥5 µm long. Likewise, fibers <0.5 µm in diameter had consistently higher PRs than fibers >0.5 µm in diameter at each corresponding sacrifice point. Day 1 concentrations of fibers >20 µm in length ranged from 1500 ± 1700 f/mg DL (Fiber 10) to 4700 ± 600 f/mg DL (Fiber 11). The PRs for Fiber 21 were higher than the average PR for the other MMVF both at 31 and 90 days; but by day 180 the PR had decreased to values close to those of the other MMVF. At 545 days, the PRs for MMVF >20 µm in length were near background level.

Conclusions:

This study showed that MMVFs are more biosoluble than crocidolite. Also, no mortality in rats was observed after short-term exposure (5 days) to MMVFs up to 545 days after last exposure.

Executive summary:

In a short-term inhalation study, Fischer 344 rats were exposed nose-only to four MMVFs (2 glass wools, 1 rock/stone wool and 1 slag wool; no mention of fibres fulfilling Note Q in this study) as well as crocidolite (positive control) at gravimetric concentrations of 30 mg/m³) for 6 hours/day for 5 days. Rats were sacrificed at 1 hour, and 1, 5, 31, 90, 180, 270, 365, and 545 days after exposure to MMVF and lung tissues were examined to assess biopersistence of the test material. Dissolution in vivo suggested as MMVF fibres ≥0.5 µm in diameter and ≥20 µm in length dissolved within 270 days post-exposure. As these decreased, the percentages observed for fibres <0.5 µm in diameter and <5 µm in length increased, which is evidence for the thicker and longer fibres dissolving or breaking over time. Very few MMVF fibres ≥20 µm in length were detected after 545 days. 26% of lung tissues examined contained any MMVF when examined at 545 days, and was almost all siliceous and with the average concentration of <200 f/mg DL. In comparison to crocidolite, MMVF dissolved or fractured in vivo whereas crocidolite fibers did not change.

Reference 9

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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: 13-week exposure was designed to determine and substantiate the maximum tolerated dose of fibrous glass for rats exposed chronically by inhalation and to establish short-term indicators that can be used to predict the maximum tolerable dose for chronic inhalation studies.
- Short description of test conditions: Fischer 344 rats were dosed (nose-only) 0.003, 0.016, 0.03, 0.045 and 0.06 mg/L of test material for 6 h/day, 5 days/week for 13 weeks
- Parameters analysed / observed: Pulmonary pathology, lung epithelium cell proliferation, lung fiber burden, lung lavage cells and chemistry

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

glass fibre

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 8 weeks
- Weight at study initiation: not specified
- Housing: Rats were housed individually in polycarbonate cages containing hardwood bedding in Hazelton 2000 chambers in rooms operated under negative pressure (-20 mm H2O))
- Diet (e.g. ad libitum): ad libitum, pelleted standard Kliba 343 rat maintenance diet (Klingentalmuche AG, Switzerland)
- Water (e.g. ad libitum): ad libitum, fresh water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each animal is supplied fresh aerosol and the air exhaled by one animal does not reach any other animal in the chamber; More details not described: method according to Sachsse et al. 1976
- System of generating particulates/aerosols: Research and Consulting Company (RCC, Geneva) fiber aerosol generation system
- Temperature, humidity, pressure in air chamber: method according to Sachsse et al. 1976 (in German)
- Air flow rate: The exposure chambers provide a positive pressure laminar flow to each animal individually
- Air change rate: method according to Sachsse et al. 1976 (in German)
- Method of particle size determination: For bivariate length/diameter analysis, a sample of each concentration of fiber glass aerosol was collected onto a Nuclepore filter. The filter was sealed in a glass bottle and stored dry until analysis. At the time of analysis, the filter surface was washed into the bottle to retain all fibers, and then filters were ashed and the ash was added back to the fiber suspension in the bottle. The suspensions were diluted to 250 mL with distilled water and dispersed by sonication. Aliquots were filtered onto 0.2-mm pore size Nuclepore membranes. After drying, the membranes were applied to scanning electron microscopy (SEM) stubs and gold coated. Bivariate length and diameter distributions were measured according to the method outlined in WHO Monograph 4 for measuring airborne man-made mineral fibers (WHO, 1985) using scanning electron microscopy (SEM). Diameters were measured at 5000x and lengths at ≥2000x, in a minimum of 20 fields and a maximum of 100 fields or 200 fiber ends, on either a JEOL T 300 SEM or JEOL 840 SEM equipped with a Videoplan Image Analysis System. Magnifications were reduced during length measurement of very long fibers so that the entire length was measured (i.e., lengths were not truncated). Counts were made of both ‘‘total fibers’’ and ‘‘WHO fibers.’’ Total fibers were defined as all particles having a length/diameter ratio ≥3. WHO fibers (i.e., respirable fibers as defined by WHO, 1985) were those fibers having a length/diameter ratio ≥3, diameter <3 µm, and length >5 µm.

- Treatment of exhaust air: not described

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol samples for the FG-exposed groups and the air-only control group were collected on filters in the same location as the animal exposure port. Sampling was performed isoaxially, directly from the output of one of the laminar flow supply tubes to eliminate isokinetic sampling bias. Fiber mass concentrations (mg fiber/m^3 air) were determined for each FG aerosol concentration once during pretest, and at least four times per week during the 13-week exposure period. For measuring fiber mass, aerosol samples were collected onto Gelman membrane filters. The number of fibers/cc air was determined at least once during pretest and once per week throughout the exposure period as follows: Samples were collected on Millipore filters, clarified by acetone vapor, placed between glass slides, and then counted using a Bausch and Lomb Balpan phase contrast microscope at a magnification of 400x. World Health Organization Monograph 4 counting rules were applied for counting the number of WHO fibers/cc (WHO, 1985) once/week. In addition to WHO fibers, the total number of fibers and nonfibrous particles was also counted on one filter every 2 weeks. Additionally, to ensure the uniformity of exposure during each 6-hr exposure period, the fiber concentrations were also monitored continuously using a RAS (MIE Inc., Bedford, MA) light scattering monitor.

- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: not described
- Composition of vehicle: air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aerosol samples were collected onto Gelman membrane filters and the number of fibers/cc air was determined at least once during pretest and once per week through exposure period. Samples were collected on Millipore filters, clarified by acetone vapor, placed between glass slides, then counted using a phase contrast microscope at a magnification of 400x. World Health Organization Monograph 4 counting rules were applied for counting the number of WHO fibers/cc once/week. In addition to WHO fibers, the total number of fibers and nonfibrous particles was also counted on one filter every 2 weeks. The fiber concentrations were also monitored continuously using a RAS light scattering monitor.

Duration of treatment / exposure:

13 weeks; then 10 weeks without aerosol exposure for recovery

Frequency of treatment:

6 hr/day, 5 days/week

Dose / conc.:

3 mg/m³ air (nominal)

Dose / conc.:

16 mg/m³ air (nominal)

Dose / conc.:

30 mg/m³ air (nominal)

Dose / conc.:

45 mg/m³ air (nominal)

Dose / conc.:

60 mg/m³ air (nominal)

Dose / conc.:

3.2 mg/m³ air (analytical)

Dose / conc.:

16.5 mg/m³ air (analytical)

Dose / conc.:

30.5 mg/m³ air (analytical)

Dose / conc.:

44.5 mg/m³ air (analytical)

Dose / conc.:

62.2 mg/m³ air (analytical)

No. of animals per sex per dose:

31 or 36 male rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: random

Positive control:

none

Observations and examinations performed and frequency:

Not described

Sacrifice and pathology:

Randomly selected animals were terminated at 7 weeks, 13 weeks, 19 weeks, and 23 weeks.
- Lung examined microscopically for pathological changes
- Lung fiber burdens were determined
- Cell proliferation in terminal airways quantified in 10-week recovery animals (23 weeks) using PCNA assay.
- Bronchoalveolar lavage analyses

Other examinations:

Lung clearance of microspheres: After 13 weeks exposure, 6 animals from each group were exposed to radiolabeled (85-Sr) microspheres by intratracheal inhalation. The animals were maintained with no further exposure for 10 weeks and whole-body radioactivity was measured weekly (or more frequently than weekly).

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal clinical signs

Mortality:

not examined

Body weight and weight changes:

not examined

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 specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No lung or pleural fibrosis observed in any exposure group

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic evidence of pulmonary change was seen at first termination time point after 7 weeks: 2/6 rats exposed to 60 mg/m^3 fibrous glass showed few small foci over surface of lung. Lesions were more exposed after 13-week exposure. These lesions were not found after 7 or 10 weeks of recovery.
Fibres and fibre fragments seen in many macrophages and almost all micro-granulomas (>3 mg/m^3); unusual clumps of macrophages in the 45 and 60 mg/m^3 exposed rats; microgranulomas found in 1/5 rats exposed to 3 mg/m^3, 4/6 rats at 16 mg/m^3, all rats in the 30, 45, 60 mg/m^3 groups. Following 13 weeks exposure and 7 weeks recovery (without exposure), the number of alveolar macrophages had decreased in all exposure groups. However, the unusual clumps of macrophages in the 45 and 60 mg/m^3 exposed rats were still evident. The number of microgranulomas had also decreased and were only observed in rats exposed to 30, 45, and 60 mg/m^3. In addition, metaplasia (bronchiolization) was observed in the epithelium lining occasional proximal alveoli in two rats in both the 45 and 60 mg/m^3 exposure groups. In this metaplasia, cells had changed from a normal squamous appearance to cuboidal. Early interstitial fibrosis was observed in one rat in both the 45 and 60 mg/m^3 exposure groups. After 13 weeks exposure followed by 10 weeks recovery, further regression in the number of pulmonary macrophages was noted, although the clumps of macrophages noted at the end of the exposure period (13 weeks) in the 45 and 60 mg/m^3 rats were still present. Microgranulomas were found in four of six rats in each of the three highest exposure groups. A minimal amount of bronchiolization was noted in one rat in each of the dose groups exposed to 16 mg/m^3 or greater. Finally a minimal amount of interstitial fibrosis was found in one rat at 16 mg/m^3, in one rat at 45 mg/m^3, and in two of six rats exposed to 60 mg/m^3.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Metaplasia (bronchiolization) observed in epithelium lining with early interstitial fibrosis observed in one rat in each of the 45 and 60 mg/m^3 exposure groups.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung cell proliferation
Lung cell proliferation was assessed by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). After 13 weeks exposure, the percentage of proliferating cells was significantly elevated (compared to positive controls) in all but the 3 mg/m^3 exposure. PCNA immunoreactive nuclei were most abundant in what appeared to be epithelial cells of the terminal bronchioles and alveolar ducts, but were also noted in alveolar macrophages, endothelial and smooth muscle vascular cells, and interstitial cells. Proliferating cells were most numerous at the bronchoalveolar duct bifurcations where fiber-induced inflammatory changes were most prominent. After the 10-week recovery period, no significantly elevated levels of lung cell proliferation were found in any fiber-exposed groups.

Details on results:

In this study, following changes in lung structure and function occurring at the higher doses could be considered: Histopathological changes in the three highest dose groups attest to chronic inflammation; these changes include an influx of phagocytic cells, microgranulomas, and minimal fibrosis. Abnormal clumps of macrophages in the lungs were observed in the 45 and 60 mg/m³ groups, suggest macrophage clearance impairment at these exposure levels. Lung toxicity was also indicated by increased total protein, LDH, NAG and neutrophils in the BAL at exposures≥16 mg/m³ as well as elevated cell proliferation, presence of cuboidal cells and metaplasia in the lung epithelium in the higher dose groups. A lung defense impairment was demonstrated by a dose-dependent retardation of microsphere clearance during the post exposure recovery phase. These changes in lung structure and functions are strong indicators that the maximum tolerated dose at 30 mg/m³ has been reached. Increasing exposure above the level of 30 mg/m³ could only be expected to lead to further overwhelming of defense mechanisms, loss of pulmonary homeostasis and induction of nonspecific lung pathology.

Dose descriptor:

other: Maximum tolerated dose for further studies

Effect level:

30 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

immunology

Critical effects observed:

not specified

Lung fiber burden

Fibers recovered from the lungs also had no significant concentration- or time-dependent dimensional differences. Lung fibers were, however, shorter and thinner than the aerosol fibers. For example, aerosol fiber arithmetic mean dimensions were 1.0 ± 0.6 µm x 21 ± 20 µm, while the lung fiber arithmetic mean dimensions were 0.6 ± 0.3 µm x 7 ± 5 µm (60 mg/m^3 concentration). The lung fiber distribution is more concentrated in the thin-short range, and the small, out-lying peaks are absent, indicating that the respiratory system has selected only the smaller-sized fibers for entry into the deep lung. In the distribution, there are substantial quantities of very long and/or very thick fibers. The number of fibers in the lung was both dose- and time-dependent. For all doses examined, lung fiber concentration increased during the 13-week exposure and decreased during the 10-week post-exposure period. The number of fibers recovered from the lungs (WHO fibers/lung) in each of the five exposure groups was directly proportional to the number of fibers in the aerosol.

Bronchoalveolar lavage

Bronchoalveolar lavage (BAL) fluid from rats after 13 weeks of aerosol exposure demonstrated dose-dependent increased in LDH and NAG activity and total protein concentration. By the 23rd week (13 weeks aerosol exposure plus 10 weeks of recovery), however, no significant dose-dependent differences were observed in any of these three parameters. Some of the cell types in the BAL demonstrated a similar effect. After 13 weeks exposure, the percentage of neutrophils and lymphocytes increased with fiber aerosol concentration. Neutrophil concentration was more than 10-fold greater in the rats exposed to 60 mg/m^3than in the air control animals: mean numbers in millions per lung were 0.07 ± 0.05 for controls, 0.54 ± 0.27 for the 30 mg/m^3exposure, and 0.78 ± 0.08 for the 60 mg/m^3exposure. Lymphocyte elevations were less striking: mean lymphocyte numbers in millions per lung ranged from 0.08 ± 0.08 in the controls to 0.287 ± 0.06 in the 60 mg/m^3exposure. After 10 weeks recovery, cell levels in all fiber groups were similar to the air control cell levels (data not shown). Macrophages constituted the majority of BAL cells (86–97%), and their absolute concentrations were similar in all dose groups at both time points (at the 13-week time point, average macrophages in millions per lung ranged from 7 ± 2 in controls to 8 ± 3 in the highest exposure; at 23 weeks the figure was 10–11 million per lung).

Conclusions:

In this study, treatment of MMVF10 by inhalation lead to effects in the lung of rats. Based on the findings in this study, a maximum tolerated dose of 30 mg/m³ can be considered for further chronic inhalation studies.

Executive summary:

In a subchronic inhalation study, MMVF10 (fibre) in air was administered to male Fischer 344/N rats (31 or 36 per dose) by nose-only exposure at concentrations of 0.003, 0.016, 0.03, 0.045, and 0.06 mg/L (nominal) for 6 hr/day, 5 days/week for 13 weeks.

The test animals were observed for 10 weeks with no exposure following the 13-week aerosol exposure. In this study, following changes in lung structure and function occurring at the higher doses could be considered: Histopathological changes in the three highest dose groups attest to chronic inflammation; these changes include an influx of phagocytic cells, microgranulomas, and minimal fibrosis. Abnormal clumps of macrophages in the lungs were observed in the 45 and 60 mg/m³ groups, suggest macrophage clearance impairment at these exposure levels. Lung toxicity was also indicated by increased total protein, LDH, NAG and neutrophils in the BAL at exposures ≥16 mg/m³ as well as elevated cell proliferation, presence of cuboidal cells and metaplasia in the lung epithelium in the higher dose groups. A lung defense impairment was demonstrated by a dose-dependent retardation of microsphere clearance during the post exposure recovery phase. These changes in lung structure and functions are strong indicators that the maximum tolerated dose at 30 mg/m³ has been reached. Increasing exposure above the level of 30 mg/m³ could only be expected to lead to further overwhelming of defense mechanisms in rat, loss of pulmonary homeostasis and induction of nonspecific lung pathology.

Reference 10

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

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/m³) 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 repeated dose toxicity data of MMVF 10 for Note Q MMVFs using a read-across approach.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal clinical signs

Mortality:

not examined

Body weight and weight changes:

not examined

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 specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No lung or pleural fibrosis observed in any exposure group

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic evidence of pulmonary change was seen at first termination time point after 7 weeks: 2/6 rats exposed to 60 mg/m^3 fibrous glass showed few small foci over surface of lung. Lesions were more exposed after 13-week exposure. These lesions were not found after 7 or 10 weeks of recovery.
Fibres and fibre fragments seen in many macrophages and almost all micro-granulomas (>3 mg/m^3); unusual clumps of macrophages in the 45 and 60 mg/m^3 exposed rats; microgranulomas found in 1/5 rats exposed to 3 mg/m^3, 4/6 rats at 16 mg/m^3, all rats in the 30, 45, 60 mg/m^3 groups. Following 13 weeks exposure and 7 weeks recovery (without exposure), the number of alveolar macrophages had decreased in all exposure groups. However, the unusual clumps of macrophages in the 45 and 60 mg/m^3 exposed rats were still evident. The number of microgranulomas had also decreased and were only observed in rats exposed to 30, 45, and 60 mg/m^3. In addition, metaplasia (bronchiolization) was observed in the epithelium lining occasional proximal alveoli in two rats in both the 45 and 60 mg/m^3 exposure groups. In this metaplasia, cells had changed from a normal squamous appearance to cuboidal. Early interstitial fibrosis was observed in one rat in both the 45 and 60 mg/m^3 exposure groups. After 13 weeks exposure followed by 10 weeks recovery, further regression in the number of pulmonary macrophages was noted, although the clumps of macrophages noted at the end of the exposure period (13 weeks) in the 45 and 60 mg/m^3 rats were still present. Microgranulomas were found in four of six rats in each of the three highest exposure groups. A minimal amount of bronchiolization was noted in one rat in each of the dose groups exposed to 16 mg/m^3 or greater. Finally a minimal amount of interstitial fibrosis was found in one rat at 16 mg/m^3, in one rat at 45 mg/m^3, and in two of six rats exposed to 60 mg/m^3.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Metaplasia (bronchiolization) observed in epithelium lining with early interstitial fibrosis observed in one rat in each of the 45 and 60 mg/m^3 exposure groups.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung cell proliferation
Lung cell proliferation was assessed by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). After 13 weeks exposure, the percentage of proliferating cells was significantly elevated (compared to positive controls) in all but the 3 mg/m^3 exposure. PCNA immunoreactive nuclei were most abundant in what appeared to be epithelial cells of the terminal bronchioles and alveolar ducts, but were also noted in alveolar macrophages, endothelial and smooth muscle vascular cells, and interstitial cells. Proliferating cells were most numerous at the bronchoalveolar duct bifurcations where fiber-induced inflammatory changes were most prominent. After the 10-week recovery period, no significantly elevated levels of lung cell proliferation were found in any fiber-exposed groups.

Details on results:

In this study, following changes in lung structure and function occurring at the higher doses could be considered: Histopathological changes in the three highest dose groups attest to chronic inflammation; these changes include an influx of phagocytic cells, microgranulomas, and minimal fibrosis. Abnormal clumps of macrophages in the lungs were observed in the 45 and 60 mg/m³ groups, suggest macrophage clearance impairment at these exposure levels. Lung toxicity was also indicated by increased total protein, LDH, NAG and neutrophils in the BAL at exposures≥16 mg/m³ as well as elevated cell proliferation, presence of cuboidal cells and metaplasia in the lung epithelium in the higher dose groups. A lung defense impairment was demonstrated by a dose-dependent retardation of microsphere clearance during the post exposure recovery phase. These changes in lung structure and functions are strong indicators that the maximum tolerated dose at 30 mg/m³ has been reached. Increasing exposure above the level of 30 mg/m³ could only be expected to lead to further overwhelming of defense mechanisms, loss of pulmonary homeostasis and induction of nonspecific lung pathology.

Dose descriptor:

other: Maximum tolerated dose for further studies

Effect level:

30 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

immunology

Critical effects observed:

not specified

Lung fiber burden

Fibers recovered from the lungs also had no significant concentration- or time-dependent dimensional differences. Lung fibers were, however, shorter and thinner than the aerosol fibers. For example, aerosol fiber arithmetic mean dimensions were 1.0 ± 0.6 µm x 21 ± 20 µm, while the lung fiber arithmetic mean dimensions were 0.6 ± 0.3 µm x 7 ± 5 µm (60 mg/m^3 concentration). The lung fiber distribution is more concentrated in the thin-short range, and the small, out-lying peaks are absent, indicating that the respiratory system has selected only the smaller-sized fibers for entry into the deep lung. In the distribution, there are substantial quantities of very long and/or very thick fibers. The number of fibers in the lung was both dose- and time-dependent. For all doses examined, lung fiber concentration increased during the 13-week exposure and decreased during the 10-week post-exposure period. The number of fibers recovered from the lungs (WHO fibers/lung) in each of the five exposure groups was directly proportional to the number of fibers in the aerosol.

Bronchoalveolar lavage

Bronchoalveolar lavage (BAL) fluid from rats after 13 weeks of aerosol exposure demonstrated dose-dependent increased in LDH and NAG activity and total protein concentration. By the 23rd week (13 weeks aerosol exposure plus 10 weeks of recovery), however, no significant dose-dependent differences were observed in any of these three parameters. Some of the cell types in the BAL demonstrated a similar effect. After 13 weeks exposure, the percentage of neutrophils and lymphocytes increased with fiber aerosol concentration. Neutrophil concentration was more than 10-fold greater in the rats exposed to 60 mg/m^3than in the air control animals: mean numbers in millions per lung were 0.07 ± 0.05 for controls, 0.54 ± 0.27 for the 30 mg/m^3exposure, and 0.78 ± 0.08 for the 60 mg/m^3exposure. Lymphocyte elevations were less striking: mean lymphocyte numbers in millions per lung ranged from 0.08 ± 0.08 in the controls to 0.287 ± 0.06 in the 60 mg/m^3exposure. After 10 weeks recovery, cell levels in all fiber groups were similar to the air control cell levels (data not shown). Macrophages constituted the majority of BAL cells (86–97%), and their absolute concentrations were similar in all dose groups at both time points (at the 13-week time point, average macrophages in millions per lung ranged from 7 ± 2 in controls to 8 ± 3 in the highest exposure; at 23 weeks the figure was 10–11 million per lung).

Conclusions:

In this study, treatment of MMVF10 by inhalation lead to effects in the lung of rats. Based on the findings in this study, a maximum tolerated dose of 30 mg/m³ can be considered for further chronic inhalation studies.

Executive summary:

In a subchronic inhalation study, MMVF10 (fibre) in air was administered to male Fischer 344/N rats (31 or 36 per dose) by nose-only exposure at concentrations of 0.003, 0.016, 0.03, 0.045, and 0.06 mg/L (nominal) for 6 hr/day, 5 days/week for 13 weeks.

The test animals were observed for 10 weeks with no exposure following the 13-week aerosol exposure. In this study, following changes in lung structure and function occurring at the higher doses could be considered: Histopathological changes in the three highest dose groups attest to chronic inflammation; these changes include an influx of phagocytic cells, microgranulomas, and minimal fibrosis. Abnormal clumps of macrophages in the lungs were observed in the 45 and 60 mg/m³ groups, suggest macrophage clearance impairment at these exposure levels. Lung toxicity was also indicated by increased total protein, LDH, NAG and neutrophils in the BAL at exposures ≥16 mg/m³ as well as elevated cell proliferation, presence of cuboidal cells and metaplasia in the lung epithelium in the higher dose groups. A lung defense impairment was demonstrated by a dose-dependent retardation of microsphere clearance during the post exposure recovery phase. These changes in lung structure and functions are strong indicators that the maximum tolerated dose at 30 mg/m³ has been reached. Increasing exposure above the level of 30 mg/m³ could only be expected to lead to further overwhelming of defense mechanisms, loss of pulmonary homeostasis and induction of nonspecific lung pathology.

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

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Conclusions:

Testing of repeated dose dermal toxicity is waived based on the fact that MMVF note Q fibres are inorganic fibres, whose physicochemical properties suggest a low potential to cross biological membranes and consequently a low potential for absorption through the skin. Furthermore, dermal exposure is not a significant exposure route for MMVF note Q fibres. MMVF note Q fibres are assessed not to possess toxic properties by repeated skin contact.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Conclusions:

Testing of repeated dose dermal toxicity is waived based on the fact that MMVF note Q fibres are inorganic fibres, whose physicochemical properties suggest a low potential to cross biological membranes and consequently a low potential for absorption through the skin. Furthermore, dermal exposure is not a significant exposure route for MMVF note Q fibres. MMVF note Q fibres are assessed not to possess toxic properties by repeated skin contact.

Additional information

Testing of repeated dose oral and dermal toxicity is waived based on the fact that MMVF note Q fibres are inorganic fibres, whose physicochemical properties suggest a low potential to cross biological membranes and consequently a low potential for absorption through the gastrointestinal tract and the skin. Furthermore, oral and dermal exposure is not a significant exposure route for MMVF note Q fibres (Adaptation from column 2 of Annex IX, 8.6.2, and Annex XI, 3). It is evaluated that systemic toxicity is unlikely after oral or dermal exposure to MMVF note Q fibres. Thus, MMVF note Q fibres are assessed not to possess toxic properties by repeated oral or dermal exposure.

Two long-term inhalation studies with exposure of male rats (Fischer 344) to up to 30 mg/m³ MMVF note Q fibres for 6h/day, 5 days/week, for 24 months showed minimal collagen deposition similar to what could be expected for any biologically inert dust at same exposure level. MMVF note Q fibres did not show any carcinogenic potential in either the lungs or in pleura, nor did the fibres induce lung fibrosis in the exposed animals. Furthermore, MMVF note Q fibres are biosoluble in the lungs fluid or in macrophages. Thus, inhalation exposure to MMVF note Q fibres does not induce local effects impairing human health. MMVF note Q fibres are inorganic fibres, whose physicochemical properties suggest a low potential to cross biological membranes and consequently a low potential for absorption through the lungs. It is evaluated that systemic toxicity after inhalation exposure to MMVF note Q fibres is unlikely. Thus, MMVF note Q fibres are assessed not to possess toxic properties by inhalation exposure.

 

Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: lung

Justification for classification or non-classification

MMVF note Q fibres are inorganic fibres, whose physicochemical properties suggest a low potential to cross biological membranes and consequently a low potential for absorption through the lungs, gastrointestinal tract and the skin. It is evaluated that systemic toxicity after inhalation, oral or dermal exposure to MMVF note Q fibres is unlikely. Long-term inhalation exposure of MMVF note Q fibres caused minimal/negligible local effects in the lungs. Furthermore, MMVF note Q fibres are biosoluble in the lungs fluid or in macrophages.

Repeated dose toxicity studies of Note Q MMVFs tested up to 30 mg/m3 in rats (determined as the MTD) revealed mild and transient effects in the lung such as increased inflammatory responses in alveolar macrophages. No fibrotic potential was seen up to this concentration. Taking a weight-of evidence approach, Note Q MMVFs do not warrant a classification for specific organ toxicity under CLP Regulation.

MMVF note Q fibres are assessed to possess no toxic properties after repeated oral, dermal or inhalation exposure, and MMVF note Q fibres shall not be classified according to the criteria in Council Directive 67/548/EEC and Regulation (EC) 1272/2008.