Glass, oxide, chemicals

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 1997 – May 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Comparable to guideline study. Applied method is based on previously published methods for analysis of test dosage, bronchoalveolar lavage with differential cell counting and protein analysis and semi-quantitative analysis of the level of cell proliferation in the lung parenchyma. The study design is not performed according to a guideline, but the method for exposure is similar to the exposure method described in EC guidelines ECB/TM/16(97) rev. 1 and ECB/TM/19(97), just as the chosen exposure concentration is similar to the concentration used for a combined long-term repeated exposure/carcinogenicity study (Searl et al. 1999 cited in end point 7.7). Not a GLP study, but methods and data are presented in a scientifically correct way.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1985

Reference 3

Reference Type:

publication

Title:

Unnamed

Year:

1999

Reference 4

Reference Type:

publication

Title:

Bromo-deoxyuridine (BrdU) uptake in the lungs of rats inhaling amosite asbestos or vitreous fibres at equal airborne fibre concentrations.

Author:

Donaldson K, Brown DM, Miller BG, Brody AR

Year:

1995

Bibliographic source:

Experimental Toxicology and Pathology, 47:207-211

Reference 5

Reference Type:

other: Guideline

Title:

Man-made mineral fibre.

Author:

Health and Safety Executive

Year:

1988

Bibliographic source:

Methods for determination of hazardous substances: 59

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male Wistar rats were exposed to whole body inhalation of aerosols of E-glass microfibre (glass fibres code 104E) at a concentration of 1000 WHO fibres/ml for 7 hours/day, 5 days/week in one of the following four dosing regimens: 1, 3, 8 or 14 days of actual exposure over a 3 week calendar period. The 8 and 14 day exposure regimens included 1 and 2 non-inhalation weekend breaks, respectively. 6-12 rats were used per dosing group (duration of exposure). The animals were sacrificed 18 hours after the final day of the 3 week test period.
The study included parallel investigations exposing rats to inhalation of other fibre types including amosite and glass microfibre code 100/475-fibres at the same concentration and in similar dosing regimen. Untreated control animals were kept in normal room air.
Following the sacrifice the lungs of the rats were subjected to bronchoalveolar lavage (BAL) and the resulting BAL fluid was subjected to analysis for cell numbers and protein analysis. For detection of proliferating cells groups of 3 rats were exposed to inhalation for 1 day and then given an intraperitoneal injection of 5'-bromo-2'-deoxyuridine (BrdU, 20 mg/kg bw) 2 hours prior to sacrifice. The proliferating cells, labelled by incorporation of BrdU during the DNA synthesis, were detected using immunohistochemistry in 5 µm histological sections cut at 6 levels throught the lung (apex to base). Data from this analysis is presented as the number of BrdU-positive cells per mm length of terminal bronchiolar/alveolar duct perimeter, as determined using an image analysis system according to a method described by Donaldson et al. (1995).

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Glass microfiber code 104E

IUPAC Name:

Glass microfiber code 104E

Constituent 2

Reference substance name:

E-glass microfibre

IUPAC Name:

E-glass microfibre

Details on test material:

- Name of test material (as cited in study report): 104E
- Produced and provided by: Manville Insulation, Mountain Technical Centre, Schuller International Inc. Littleton, Colorado, USA.
- Substance type: Man-made vitreous fibre
- Physical state: Solid, respirable fiber material (dust)
- Composition of test material, percentage of components: See Table 1.
- Fiber geometric dimensions (length, diameter and surface area, mean values): See Table 2
- Lot/batch No.: NA.
- Stability under test conditions: Stable
- Other: Test material provided in a bale. Respirable fibres were created by chopping in a Manesty rotary chopper, followed by milling in a Retsch Pin mill (Type ZM1) fitted with a 1 mm mesh.
- Analysis and characterisation of E-glas microfibre test fibres was performed by scanning electron microscopy (SEM) counting fibre numbers and measuring fibre lengths and diameters according to methods proposed by WHO-EURO (World Health Organisation, 1985). Fibres were defined to have a length:diameter ratio of >3:1 and a minimum length of 0.4 µm. Data recorded for the fibre type were field number, length, diameter (maximum), and number of fibre ends in the field. The uncertainty of repeated SEM analysis of fiber dimensions was ± 13% for length and ± 0.09µm for diameter measurements. For the statistical analysis, the number of fibres was accounted for according to length categories (<5 µm; 5-10 µm; 10-15 µm; 15-20 µm, and > 20 µm).

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Inhalation exposure: Groups of 6-12 animals were exposed to 1000 fibres/ml air in whole body exposure chambers for 7 hours per day,5 days/week in one of the following four dosing regimens: 1, 3, 8 or 14 days of actual exposure over a 3 week calendar period. The 8 and 14 day exposure regimens included 1 and 2 non-inhalation weekend breaks, respectively. 6-12 rats were used per dosing group (duration of exposure). The animals were sacrificed 18 hours after the final day of the 3 week test period. When not in the exposure chamber, animals were kept in conventional cages.
For detection of proliferating cells groups of 3 rats were exposed to inhalation for 1 day and then given an intraperitoneal injection of 5'-bromo-2'-deoxyuridine (BrdU, 20 mg/kg bw) 2 hours prior to sacrifice.

IN-LIFE DATES:
From: Age at study initiation unknown
To: Animals were sacrificed 18 hours after the last exposure day (1 day or 3 calendar weeks)..

- Source: Not described
- Age at study initiation: Not described
- Weight at study initiation: Not described
- Fasting period before study: Not described
- Housing: Not described
- Diet (e.g. ad libitum): Not described
- Water (e.g. ad libitum): Not described
- Acclimation period: Not described

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not described
- Humidity (%):Not described
- Air changes (per hr): Not described
- Photoperiod (hrs dark / hrs light): Not described

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

PREPARATION OF FIBRE AEROSOLS:
Dust clouds were generated in 1.3 m^3 chambers using a Timbrell Dust Feeder ( fitted with glass linings). The respirable fibre concentrations in the exposure chambers were determined gravimetrically using a Casella MRE 113A. Fibre concentrations were monitored by hourly counts (PCOM) of a short period (1 minute) 0.2 liter samples, 8 per exposure day following procedures described in Method for Detection of Hazardous Substances 59, Health and Safety Executive (1988). The respirable fibre mass concentrations were measured daily and a fibre number to mass ration was determined using PCOM every 2 weeks. The size distribution of fibres in the exposure air was determined by SEM performed on short period samples collected on polycarbonate filters.

VEHICLE
- Justification for use and choice of vehicle : athmospheric air, which is a reasonable vehicle for inhalation aerosols.
- Concentration in vehicle: 1000 WHO fibres/ml.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Fibre concentrations were monitored by hourly counts (PCOM) of a short period (1 minute) 0.2 liter samples, 8 per exposure day following procedures described in MDHS 59, Health and Safety Executive (1988). The respirable fibre mass concentrations were measured daily and a fibre number to mass ration was determined using PCOM every 2 weeks.
The size distribution of fibres in the exposure air was determined by scanning electron microscopy (SEM) performed on short period samples collected on polycarbonate filters. The SEM fibre counting and measurement was performed following validated procedures documented in the UK Accreditation Service (UKAS) quality system (EN 45001). Fibre measurements were performed with a Hitachi S520 SEM or a Cambridge S250 MkII SEM at a magnification of 5000X. The measurements were performed after calibration against a grid calibrated by the National Physical Laboratory, and analysis by counting of fibres and measurement of length and diameter was performed in accordance to the methods described by WHO-EURO (World Health Organisation, 1985).

Duration of treatment / exposure:

7 hours per day for 1 day, 3 days, 8 days or 14 days of actual exposure over a max. period of 3 calendar weeks. The 8 and 14 day exposure regimens included 1 and 2 non-inhalation weekend breaks, respectively.

Frequency of treatment:

See above.

Doses / concentrations

Remarks:

Doses / Concentrations:
1000 WHO fibres/ml
Basis:
analytical conc.

No. of animals per sex per dose:

6-12 male animals per dosing group (time interval)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The selection of protocol and dose was based on data from previous studies on short term and chronic inhalation of fibres (mainly asbestos) in earlier studies by Institute of Occupational Medicine and data provided by the Joint European Medical Research Board (JEMRB) on fibre concentrations and size distributions.
- Rationale for animal assignment: Random

Positive control:

The study included parallel investigations exposing rats to inhalation of other fibre types including amosite and glass microfibre code 100/475-fibres at the same concentration and in similar dosing regimen.

Examinations

Observations and examinations performed and frequency:

BRONCHOALVEOLAR LAVAGE (BAL): After sacrifice of the animals the thoracic cavity was opened, the trachea cannulated and the lungs were lavaged with four 8 ml aliquots of saline at 37 °C. The resulting BAL fluid was pooled and centrifuged for recovering the cells from the fluid. the cells were resuspended in F-10 medium (Gibco, Paisley) containing 0.2% bovine serum albumin. Total cell counts were made and cytocentrifuge smears were prepared and stained with Diffquick (Merz Dade, Switzerland) to allow for differential cell counts. The cell-free portion of BAL fluid from the first 8 ml aliquot was used to determin the protein concentration by standard spectrophotometric assay (Biorad Laboratories, Munich, Germany) Bovine serum albumin was used to create a standard curve. Standards and test samples were analysed in triplicate at 595 nm, and the protein concentration (in mg/l) in the samples were calculated from the standard curve using linear regression.

CELL PROLIFERATION USIN BRDU DNA LABELLING: For detection of proliferating cells groups of 3 rats were exposed to inhalation for 1 day and then given an intraperitoneal injection of 5'-bromo-2'-deoxyuridine (BrdU, 20 mg/kg bw) 2 hours prior to sacrifice. The proliferating cells, labelled by incorporation of BrdU during the DNA synthesis of mitosis, were detected using immunohistochemistry in 5 µm histological sections cut at 6 levels throught the lung (apex to base). Data from this analysis is presented as the number of BrdU-positive cells per mm length of terminal bronchiolar/alveolar duct perimeter, as determined using an image analysis system according to a method described by Donaldson et al. (1995).

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: No

HAEMATOLOGY: No.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: No data

Results and discussion

Results of examinations

Details on results:

BRONCHOALVEOLAR LAVAGE: Dosage/Exposure duration - dependent increases in the total cell number in BAL fluid, the fraction of granulocytes in the BAL fluid and the total concentration of protein in the BAL fluid.

CELL PROLIFERATION USING BRDU DNA LABELLING: Increased numbers of proliferating cells in the alveolar bronchioles in animals exposed 1 day (7 hours) by inhalation of E-glass microfibre (104E) fibres.

Effect levels

Dose descriptor:

NOAEC

Remarks:

Only one concentration tested - Dosage scheme related to the accumulated exposure time

Effect level:

1 000 other: WHO fibres/ml

Sex:

male

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Results of assessments from bronchoalveolar lavage: total cell numbers, % granulocytes, and protein concentrations as means and estimated standard errors (s.e).
	Length of inhalation exposure
Fiber type	0 days		1 day		3 days		8 days		14 days
	Mean	s.e.	Mean	s.e.	Mean	s.e.	Mean	s.e.	Mean	s.e.
Total cell number in BAL (*10^6cells)
No fibre	6.2	0.5
104E			7.1	1.8	5.5	1.4	7.8	1.9	11.9	3.0
% granulocytes in BAL
No fibre	0.4	0.2
104E			2.2	1.7	5.1	3.4	6.0	3.9	8.9	5.4
Protein concentration in BAL (units/ml)
No fibre	144	12
104E			162	29	175	31	258	46	253	45

Table 2:Numbers of BrdU-positive cells at six levels from apex to base of the lungs of rats after inhalation of fibres for 7 hours (1 day). Table shows mean and standard errors of inter-individual data.
	Level in lung (Apex = 1, Base = 6), Data in counts per mm duct
	1		2		3		4		5		6		Average
Fibre type	Mean	s.e.	Mean	s.e.	Mean	s.e.	Mean	s.e.	Mean	s.e.	Mean	s.e.	Mean	s.e.
No fibre	0.81	0.35	0.70	0.28	1.04	0.32	1.04	0.29	0.46	0.16	0.57	0.26	0.74	0.13
104E	2.63	0.68	3.20	0.66	3.42	0.57	1.12	0.34	1.77	0.46	0.57	0.40	2.04	0.21

Applicant's summary and conclusion

Conclusions:

Rats were exposed to inhalation of E-glass microfibre (code 104E) fibres for 7 hours per day for either 1, 3, 8 or 14 days of actual exposure over a period of max. 3 calendar weeks. Following sacrifice the lungs, the BAL fluid was examined for total cell count, the fraction of granulocytes and the total concentration of proteins. This analysis showed a progressive increase in the total cell number, the fraction of granulocytes and in the total protein concentration with increased duration of the accumulated, repeated exposure. The data indicate induction of inflammatory response even after just one day of 7 hours of exposure. Further, analysis of the number of proliferating cells per mm bronchiolar duct was investigated using BrdU DNA labelling revealed significantly increased numbers of proliferating cells in the lungs of animals exposed to E-glass microfibre (statistically significant at p<0.05, compared to non-treated controls). This is also indicative of an inflammatory response in the lung parenchyma. In conclusion the study data indicate that inhalation of E-glass microfibre is able to induce an inflammatory response in the lungs of rats after a single or 3 to 14 days of repeated exposure .

Executive summary:

Rats were exposed to inhalation of E-glass microfibre (code 104E) fibres for 7 hours per day for either 1, 3, 8 or 14 days of actual exposure over a period of max. 3 calendar weeks. Following sacrifice the lungs, the BAL fluid was examined for total cell count, the fraction of granulocytes and the total concentration of proteins. This analysis showed a progressive increase in the total cell number, the fraction of granulocytes and in the total protein concentration with increased duration of the accumulated, repeated exposure. The data indicate induction of inflammatory response even after just one day of 7 hours of exposure. Further, analysis of the number of proliferating cells per mm bronchiolar duct was investigated using BrdU DNA labelling revealed significantly increased numbers of proliferating cells in the lungs of animals exposed to E-glass microfibre (statistically significant at p<0.05, compared to non-treated controls). This is also indicative of an inflammatory response in the lung parencyma. In conclusion the study data indicates that inhalation of E-glass microfibre is able to induce an inflammatory response in the lungs of rats after single or 3 to 14 days of repeated exposure .