Potassium aluminium silicate amorphous glass fibres

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: ECB/TM/16/97

Deviations:

yes

Remarks:

only highest dose used, more endpoints were studied and animals were followed up for a longer period

Principles of method if other than guideline:

only highest dose used, more endpoints were studied and animals were followed up for a longer period (12 months rather than 90 days)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

amporphous glass fibre formed from the melting and fiberisation of predominately slilcon dioxide, calcium oxide, magnesium oxide

EC Number:

610-130-5

Cas Number:

436083-99-7

Molecular formula:

Amorphous glass consisting of SinO(3n-1)2(n-1) Polymeric anions ionically bonded to Ca2+ and Mg2+ cations or other alkaline earth cations

IUPAC Name:

amporphous glass fibre formed from the melting and fiberisation of predominately slilcon dioxide, calcium oxide, magnesium oxide

Test material form:

solid: fibres

Details on test material:

exposure was to a test article prepared from commercial high purity AES wool, non-commercial AES fibre containing 5% Zirconia and commercial RCF, prepared by milling, grinding and size separation
- Name of test material (as cited in study report): Fibre A (Commerical high purity AES Wool), Fibre B (non-comerical AES fibres containing 5% zirconia) and Fibre D (RCF)
- Substance type: milled white fibre
- Physical state: Solid
- Other: test article sizing: detailed in table 1 in the additional materials section as the activity of the fibres is determined to a large part on their size

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation: 9-10weeks
- Weight at study initiation: 110-120g
- Housing: 2 rats per polycarbonate cage
- Diet (e.g. ad libitum): Commerce chow Atromin N 1324 special prepared ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Two weeks before tube training

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 55 +/- 15
- Air changes (per hr): Not recorded; varied with temperature and humidity controls
- Photoperiod (hrs dark / hrs light): 12h light dark cycle

IN-LIFE DATES: August 15th 1997 (initiation) exposure completed December 14th 1997

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Fibre size not calculated as MMAD.
for Fibre A size distribution of aerosol 50% fibres were less than 10.4 microns long, 50% less than 1.25 micron diameter 50% had aerodynamic diameter of less than 4.0 microns.
for Fibre B size distribution of aerosol 50% fibres were less than 11.6 microns long, 50% less than 1.22 micron diameter 50% had aerodynamic diameter of less than 4.0 microns.
For fibre D size distribtion of aerosol 50% fibres were less than 11.0 microns long, 50% less than 1.35 micron diameter 50% had aerodynamic diameter of less than 4.3 microns.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow past system as described in Bernstein DM, Thevenaz P, Fleissner H, Anderson R,Hesterberg TW, Mast R 1995 Evaluation of the oncogenic potential of man-made vitreous fibers: the inhalation model Ann Occup Hyg 1995; 39(5):661-72 except that the aerosol was generated using a pneumatic disperser, static on the aerosol was neutralised using a Nickel 63 radiation source
- Method of holding animals in test chamber: Battelle Tubes
- Air flow rate: 1 litre/min
- Method of particle size determination: samples of the aerosol were collected on membrane filters , these were weighed for gravimetric analysis and fibres counted by SEM, except for Amosite positive control TEM was used.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the aerosol were collected on membrane filters, these were weighed for gravimetric analysis and fibres counted by SEM, except for Amosite positive control where TEM was used.
Fibre A - 644 ± 176 WHO fibres/ml
152 ± 48 fibres longer than 20µm /ml
Mass concentration 71 ± 17.6 mg/m3

Fibre B - 539 ± 159 WHO Fibres/ml
152 ± 40 fibres longer than 20µm /ml
Mass concentration 49.8 ± 8.4 mg/m3

Fibre D - 515 ± 139 WHO Fibres/ml
137 ± 30 fibres longer than 20µm /ml
Mass concentration 58.6 ± 10.4 mg/m3

Further details given below

Duration of treatment / exposure:

total 89 days

Frequency of treatment:

6h/day, 5days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Fibre A - 644 ± 176 WHO fibres/ml 153 ± 48 fibres longer than 20µm /ml Mass concentration 70 ± 17.6 mg/m3
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Fibre B 539 ± 159 WHO Fibres/ml 152 ± 40 fibres longer than 20µm /ml Mass concentration 49.8 ± 8.4 mg/m3
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
Fibre D - 515 ± 139 WHO Fibres/ml 137 ± 30 fibres longer than 20µm /ml Mass concentration 58.6 ± 10.4 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

57 male

Control animals:

yes, concurrent no treatment

Details on study design:

The EU guideline ECB/TM/16(97) rev. 1 was largely followed except that some animals were allowed to survive for one year after exposure for 90 days to enable any pathology to resolve or develop. Cell proliferation in the terminal bronchioles and in the lung parenchyma was measured by the BrDU method. The dose of fibres in the lung was estimated by counting the fibres in the ashed tissue. RCF fibres were counted by SEM and amosite by TEM Non fibrous aluminosilicate glass content was measured using chemical analysis for the aluminium content of the ash, The dust content of the lung associated lymph nodes was also measured. The integrity of macrophage clearance from the lungs was estimated by thoracic radioactivity measurements after a brief inhalation of tracer particles.
- Dose selection rationale: to ensure comparable dosing of long fibres with positive control
- Rationale for animal assignment (if not random): Randomly assigned by body weight using Datatox system so that mean weight of groups was within 1% of overall mean

Positive control:

amosite asbestos long fibre dose 756 ± 133 WHO fibres/ml
146 ± 28 fibres longer than 20µm /ml
Mass concentration 6.5 ± 0.9 mg/m3

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Up to three months Individual body weights weekly to nearest 0.1g and once every two weeks for the duration of study

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

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
on lung lavage fluid

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: cellular content of lung lavage fluid determined on sacrifice

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: Yes (see table)

Other examinations:

clinical chemistry and cellular content, on lung lavage fluid

Fibre and particle content of lungs and lung associated lymph nodes

Statistics:

Differences between groups were considered statistically significant at p <0.05. Data were analysed using analysis variance. if the group means differed significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups based on the Dunnett's test. The analysis of the data was done with SAS software package (version SAS Institute, Cery, NC USA, Release 6.12 on DEC Alpha 2000 4/233).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

in lung lavage fluid

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

small increase in lung weight

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

alvveolar and interstitial deposits of fibre or particle laden macrophages

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
- no clinical abnormalities were observed and no animals died during the study period

BODY WEIGHT AND WEIGHT GAIN
- There was no effect of AES on body weight but exposure to non-fibrous aluminosilicate particles and amosite asbestos resulted in a significant (P<1% - Dunnett’s test) reduction in body weight and a significant increase in lung weight. The body weight reduction with amosite occurred on days 53-91.

CLINICAL CHEMISTRY: LUNG LAVAGE FLUID,
- no effects on LDH or beta-Glucuronidase, small increase in total protein initially but had recovered after 1.5 months.

ORGAN WEIGHTS
- Fibres A & B caused a small increase in lung weight. Fibres A & B, unlike the other three test articles did not cause a significant increase in lung associated lymph node weights.

GROSS PATHOLOGY
- none observed

HISTOPATHOLOGY: NON-NEOPLASTIC
Macroscopic findings - None detected
Microscopic findings
All test materials showed very slight to slight alveolar and interstitial deposits of fibre or particle laden macrophages. This declined by 12 month in the AES exposed group. A summary of fibrosis scores is reported table 9. All other effects were slight or very slight and more intense in RCF and Amosite asbestos.
- Mean Wagner fibrosis grade: Fibre A - 2.2 after 4 days and 3.0 after 12 months
Fibre B - 3.0 after 4 days and 3.0 after 12 months

OTHER FINDINGS
- Cell proliferation in the terminal airways and lung parenchyma was studied using BrdU assay, no lasting effect was observed. The proportion of dividing cells increased after all exposure but for RCF and amosite asbestos this effect was greater in the terminal airway than in the lung parenchyma. The effect of Fibres A & B were only significant immediately after exposure but the effect of amosite persisted for the entire 12 month period of study,

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 535 other: WHO f/ml

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 3 Terminal Body weights (SD) male rats(g)

Control	Amosite asbestos	Fibre A	Fibre B	Fibre D	Non fibrous aluminosilicate
259.0 (17.4)	235.1 (11.8)	259.3 (8.5)	250.3 (20.9)	251.9 (22.6)	250.0 (8.7)

 

Table 4 Terminal lung weights

Control	Amosite asbestos	Fibre A	Fibre B	Fibre D	Non fibrous aluminosilicate
1.049 (0.080)	1.214  (0.031)	1.135 (0.13)	1.134 (0.092)	1.188 (0.087)	1.425 (0.096)

Figures in bold 

Table 5 Clearance of test substance from the lung Mean (90%CL) days

Material	HALF TIME IN DAYS
	Number of fibres	Number of WHO fibres	Number of fibres >20 microns long	Mass
Fibre A	47 (25-68)	44 (27-62)	15 (12-19)	44 (29-58)
Fibre B	102 (84-120)	90 (74-106)	50 (43-58)	74 (62-85)
Fibre D	338 (286 – 389)	328 (269 – 388)	145 (120 – 170)	145 (120 – 170)
Amosite	402 (270-536)	502 (298-706)	561 (174-948)	444 (295-594)
Non fibrous aluminosilicate glass				91 (79-102

Half times calculated according to ECB/TM/26 counts less than 5% of initial burden discounted.

 

Table 6 Lung contents of particle and fibres (LALN = lung associated lymph nodes)

Fibre	Sacrifice [Months]	WHO Fibres [10^6/lung]		Fibres (L>20μm) [10^6/lung]		Calculated mass [mg]
						Fibres		Particles
		Lung	LALN	Lung	LALN	Lung	LALN	Lung
Fibre A	0.1	38.2	0.009	1.760	0.000	0.609	0.000
	0.5	19.0	0.011	0.499	0.000	0.289	0.000
	1.5	13.9	0.018	0.160	0.000	0.224	0.000
	3	9.8	0.041	0.042	0.000	0.133	0.001
	6	9.2	0.063	0.010	0.000	0.124	0.001
	12	2.5	0.063	0.001	0.000	0.041	0.001
Fibre B	0.1	31.1	0.005	3.82	0.002	0.669	0.000
	0.5	20.2	0.008	2.12	0.002	0.420	0.000
	1.5	23.0	0.015	1.30	0.003	0.429	0.000
	3	10.4	0.009	0.55	0.001	0.188	0.000
	6	5.0	0.016	0.23	0.001	0.085	0.000
	12	2.0	0.019	0.05	0.000	0.035	0.000
Fibre D	0.1	38.3	4.8	7.90	2.13	1.207	0.239
	0.5	36.4	3.1	7.00	0.82	1.161	0.084
	1.5	41.1	6.8	7.92	0.37	1.175	0.265
	3	32.2	5.2	6.17	1.11	0.813	0.097
	6	22.8	5.2	3.15	1.08	0.494	0.259
	12	18.2	2.0	1.55	0.32	0.222	0.034
Amosite	0.1	145.7	0.076	14.74	0.005	1.053	0.001	-
	0.5	133.2	0.093	14.19	0.015	0.869	0.002	-
	1.5	139.2	0.082	17.35	0.008	1.012	0.001	-
	3	110.1	0.206	12.66	0.005	0.846	0.002	-
	6	93.2	0.135	12.13	0.004	0.700	0.002	-
	12	88.8	0.149	10.82	0.000	0.619	0.001	-
Non fibrous aluminosilicate	0.1							4.60
	0.5							3.93
	1.5							4.05
	3							2.96
	6							1.00
	12							0.33

* For the NF Particulate the mass was determined by chemical analysis

Table 7 Clearance of Tracer particles from alveolar region of lung

Materials	Half time of alveolar tracer clearance (95 CL) days
	5 days post exposure	6 months post exposure
Control	56 (54-58)	66 (60-73)
Fibre A	88 (79-98)	86 (79-95)
Fibre B	69 (64-75)	67 (63-72)
Fibre D	435 (343 – 594)	467 (314 – 908)
Nonfibrous aluminosilicate	1202 (778-2641)	infinite
Amosite asbestos	125  (112-140)	81 (75-89)

All clearance times significantly slower than control at p<0.001

RCF, amosite and AES and the non-fibrous aluminosilicate glass deposited in the lung at concentrations that overloaded lung clearance. But the effect of the non-fibrous materials is much more extreme.

Table 8 CellProliferationindex

Location	Material	Proliferation index (%) at two times after exposure ceased
		At end of exposure		12 months after end of exposure
		Mean	SD	Mean	SD
Terminal bronchiolus	Control	1.20	0.54	0,77	0.17
	Fibre A	2.14	0.66	0.81	0.18
	Fibre B	2.63	0.86	1.09	1.00
	Fibre D	7.02	3.74	0.93	0.07
	Amosite	12.75	2.56	3.47	1.17
	NFP	4.78	1.79	0,94	0.23
Lung parenchyma	control	1.64	0.65	2.65	0.97
	Fibre A	2.19	1.01	2.60	0.25
	Fibre B	2.28	0.59	3.18	0.85
	Fibre D	4.11	1.53	4.36	0.16
	Amosite	4.64	1.17	4.26	0.79
	NFP	4.47	2.18	3.53	1.17

The effects of amosite in the terminal airway are significantly different (p<0.001) throughout the study.

Table 9 Mean Wagner Fibrosis Grades and grade involving at least 4% of lung parenchyma at 4 days and 12 months post exposure 

Material	4 days		12 months
	Mean Wagner grade	Grade 4% of total parenchyma	Mean Wagner grade	Grade 4% of total parenchyma
Control	1	-	1.33	-
Fibre A	2.2	-	3.0	-
Fibre B	3.0	-	3.0	-
Fibre D	3.0	-	3.75	0.72
Amosite	4.0	2.16	4.0	2.57
Non fibrous aluminosiliicate	3.0	-	4.0	2.41

Table 10: Biochemicalparametersin lung lavage fluid, normalised to control = 1.

	Months\after end of exposure	LDH	BETA glucuronidase	Total protein
control	ALLTIMES	1.0	1.0	1.0
Fibre A	0	1.31	1.08	1.43
	1.5	1.35	1.13	1.15
	3	1	0.92	1.10
	6	1	1.08	1.07
	12	0.85	0.83	0.99
Fibre B	0	1.42	1.33	1.54
	1.5	1.19	0.96	0.97
	3	0.90	0.75	0.98
	6	1.00	1.00	1.08
	12	1.71	1.17	1.12
RCF	0	3.58	3.92	2.90
	1.5	2.95	2.08	1.95
	3	2.55	2.92	2.05
	6	1.9	1.92	1.67
	12	1.69	1.38	1.52
Amosite	0	2.62	2.93	1.90
	1.5	2.27	1.54	1.45
	3	1.93	2	1.72
	6	1.81	1.75	1.70
	12	1.69	1.38	1.63
NFP	0	10.35	24.25	5.70
	1.5	10.69	14.29	4.37
	3	8.93	23.83	4.09
	6	6.45	15.00	3.09
	12	3.46	3.5	2.18

Figure in bold are significantly different from control p<0.01

Applicant's summary and conclusion

Conclusions:

Low biopersistence fibres such as the substance under discussion produce no significant pathological response when inhaled at very high concentrations.

Executive summary:

In this 90 day study two AES fibres were examined alongside RCF and amosite fibres as a positive control. The AES fibres in contrast to the other fibres did not display any significant pathological response and no deterioration of the lung clearance mechanism was observed. The MTD used for this study is in excess of 500 times greater than the recommended exposure levels for AES fibres in EU countries, which is much greater than the normal safety margins applied for workplace substances.

READ ACROSS INFORMATION:

In accordance with REACH Annex XI, paragraph 1.5: Grouping of substances and read across approach, Alkaline Earth Silicate fibres (REACH registration01-2119457644-32-0000)have been identified as an analogue substance where adequate testing already exists.

Man Made Vitreous Fibres (MMVF) are UVCB substances classified as CLP category 1a or 2 carcinogens depending on their content of various elements, both Potassium Alumino Silicate Fibres and Alkaline Earth Silicate Fibres have compositions placing them in category 3. Note Q of Directive 97/69/EC adapting Council Directive 67/548/EEC further exempts these fibres from labelling as carcinogens as they satisfy one or more of the test criteria listed. They share this exemption with many other vitreous fibres in use as insulation products and many examples have been tested by all available routes. 

Potassium Alumino Silicate fibres are made using an identical manufacturing process and have the same range of fibre diameters as the well established Alkaline Earth Silicate fibres. It follows that respirable dust is present in the workplace in similar quantities and with similar dimensions. The two substances differ in that the with Alkaline Earth Silicate fibres, Calcium and Magnesium are added to the glass to promote bio-solubility whereas with Potassium Alumino Silicates fibres, the same objective is achieved by the addition of Potassium. The soluble elements are added in sufficient quantity to satisfy the 18% rule inNote Q, which allows the use of in-vivo testing to demonstrate low bio-persistence and exoneration from carcinogen classification.

Alkaline Earth Silicate fibres have been tested by 90 day and 2 year inhalation studies as well as the short term biopersistence tests required in Note Q. Potassium Alumino Silicate fibres have also been tested for their biopersistence after Intratracheal Installation in rats and have satisfied the requirement of having a half life of <40 days. Unless some toxic element were present there is a consensus that fibres with similar bio-persistence will display similar toxicological behaviour when inhaled. The inorganic materials used in the production of Alkaline Earth Silicate Fibres are the oxides of Calcium, Magnesium and Silicon. The inorganic materials used in the production of Potassium Alumino Silicate fibres are the oxides of Potassium, Aluminium and Silicon. All of these are ubiquitous in the environment and are common as the mineral content in foodstuffs. It is therefore not feasible that the chemical difference between these fibres will result in a different toxicological result.

Potassium Alumino Silicate fibres contain a minor addition of Zirconium Oxide. This is also used optionally in the production of Alkaline Earth Silicate fibres. Fibre B in the study report, “Subchronic Inhalation Study with High Temperature Insulation Fibres in Rats” was an Alkaline Earth Silicate fibre containing 5% Zirconia addition. Since fibre B gave very similar results to Fibre A (Alkaline Earth Silicate with no Zirconia addition) it is concluded that 6% addition of Zirconia has no effect on the end points investigated in this study.

The report, “In vitro solubility of Alkaline Earth and Potassium Alumino Silicate Fibres” compares the solubility of these two fibre types in simulated alveolar conditions. Both display similar levels of solubility adding extra confidence to the reliability of read across in inhalation testing.

In summary the results from existing 90 day and 2 year inhalation studies for Alkaline Earth Silicate fibres linked to the short term bio-persistence study with the Potassium Alumino Silicate fibres provide reliable evidence as to the lack of toxic potential for Potassium Alumino Silicate fibres. This is supported by the lack of effect on the lungs of the rats used in the biopersistence studies. With this background we do not believe that further animal testing can be justified.