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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 15 Oct. 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published data not containing full information available in the original study report
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and regional lymph nodes. Post-exposure recovery period up to one year.
Principles of method if other than guideline:
Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline silica).
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Silicon dioxide
EC Number:
231-545-4
EC Name:
Silicon dioxide
Cas Number:
7631-86-9
Molecular formula:
O2Si
IUPAC Name:
silicon dioxide (amorphous)
Details on test material:
- Name of test material (as cited in study report): Aerosil 200:
CAS-Name: Silica, amorphous, fumed, crystalline-free,
CAS-No.: 112945-52-5
- Substance type: inorganic
- Physical state: solid
- Surface area (BET): 151 m2/g (Report p. 59 Specification Certificate)
- Analytical purity: >99.8 % (SiO2)
- Particle size: The range of the geometric agglomerate/aggregate size distribution was 1  to about 120 µm for the amorphous silicas with maxima at approx. 10 µm and 100 µm 
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO
- Age at study initiation: 6 weeks
- Weight at study initiation: males 107 +/- 1 g - 109 +/- 2 g; females 105 +/- 1 g - 109 +/- 1 g
- Fasting period before study: no
- Housing: single in stainless steel wire cages during exposure. 5 males/5 females/ stainless steel cage after the exposure period
- Diet: no access during exposure
- Water:unflouridated tap water ad libitum (no access during exposure)
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-1
- Humidity (%):65 - 75
- Air changes (per hr): 12x/h (airflow approximately 40 m3/h)
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Remarks on MMAD:
MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton H 1000 inhalation chambers
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator composed of a dust feed mechanism and an atomizer operated by compressed air
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C/65 - 75 % rel. humidity
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: Primary particle size calculated using arithmetic mean of transmission electron micrograph magnifications. These particles form agglomerates and aggregates for which it was not possible to determine the aerodynamic size distribution in the test atmosphere due to the weakness of the bonds and the electrostatic charge fo the particles.
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetry - Air samples are drawn through glass fiber filters (Sartorius SM 13430) and weighed (3 - 4 times per day)
- Samples taken from breathing zone: no data


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the test material in the test atmospheres were determined by gravimetry. Samples of the test atmospheres were drawn through glass fibre filters (Sartorius SM 13430). The filters were weighed just before and after sampling. No further details are available in the literature paper.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1.3, 5.9 or 31 mg/m3 (mean analytical values)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1, 6 and 30 mg/m3 (target concentrations)
Basis:
nominal conc.
No. of animals per sex per dose:
70 animals/sex/dose
Aerosil 200: assigned dose groups B, C, and D, each sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 
Control animals:
yes
Details on study design:
- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks
Positive control:
Quartz (crystalline silica, 58 mg/m3) included (assigned Group G)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery


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
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females



URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes



NEUROBEHAVIOURAL EXAMINATION: No


OTHER: ---
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung
Other examinations:
Relative organ weights
Statistics:
Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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 examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
Respiration rate: concentration-related increase
No mortality

BODY WEIGHT AND WEIGHT GAIN
No effect in females at all dose levels
Depressive effect in males:
1 mg/m3: slightly at day 14 of exposure only (~ -5%)
6 mg/m3: slightly from day 49 to day 77 of exposure (~ - 6 to <5 %)
no more significant by end of exposure (day 91)
30 mg/m3: significantly throughout exposure: ~ -7 - -10 %, day 91: -7 %
Recovery: no difference from control at day 455 (52 weeks post-exposure)


HAEMATOLOGY
1 mg/m3: no effects
6 mg/m3: White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
but concentration-response relationship was poor.
After 3 months recovery, these blood parameters normalized in males and females.
30 mg/m3: Red blood cell count and hemoglobin were statistically higher in males, but not in females.
White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
at 3 months of recovery (days 176/177), but concentration-response relationship was poor.
In females, a slight increase above the control group apparently still existed after 6 months of recovery (day 275).


CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, thymus, adrenals, testes, brain, spleen, kidney
Treatment-related degrees of severity: swollen lungs and enlarged mediastinal lypmph nodes at the end of exposure
LUNG
1 mg/m3: no significant increase
6 mg/m3: mean increase in relative weight 1.7x (males), 1.4x (females)
30 mg/m3: mean increase in relative weight 2.3x (males), 2.0x (females)
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes, the degree of severity being treatment-related.
At 6 and 30 mg/m3, collagen content in the lungs was clearly increased, most pronounced in males.

The above-mentioned effects gradually subsided after the exposure period,
but in males exposed to 6 and 30 mg/m3 the collagen content was still above control values at the end of the study.


HISTOPATHOLOGY: NON-NEOPLASTIC
Accumulation of alveolar macrophages and granular material, cellular debris, polymorphonuclear leucocytes, increased septal cellularity.
Alveolar bronchialisation, focal interstitial fibrosis, cholesterol clefts and granuloma-like lesions in the lung.
The granuloma-like lesions were seen in a few animals at the end of exposure period and after 13 weeks of recovery. They did not show fibroblastic activity and hyalinization and regressed during recovery [not progressive, i.e. no silicogenic nodules formed (no silicosis)].

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as focal necrosis and slight atrophy of the olfactory epithelium.

Interstitial fibrosis was not noted directly after the exposure period, but appeared with a delay,
for the first time observed after 13 wks post-exposure: increasing incidence especially in 30-mg rats, and a few in the 6-mg group,
but decreased in severity and frequency until the end of the study (Report p. 51).

All types of pulmonary lesions were more marked in males than in females.


The level of 1.3 mg/m3 induced only slight changes after 13-wk exposure, which generally recovered quickly (no differences from control after 13-wk post-exposure).

Morphological changes after 13-wk exposure, that were considered statistically significant at 1.3 mg/m3:

males females ! males females
treated ! untreated controls
------------------------------------------------------------------------------------------------------------------------------
Accumulation of alveolar macrophages: slight in 10/10 (very) slight in 10/10 ! (very) slight 4/10 slight in 1/10
Intra-alveolar polymorphonuclear !
leukocytes: (very) slight in 6/10 (very) slight in 8/10 ! 0/10 0/10
Increased septal cellularity: ( very) slight in 10/10 (very) slight in 9/10 ! very slight 1/10 very slight 1/10
Olfactory epithelial atrophy: (very) slight in 5/10 (very) slight in 8/10 ! 0/10 0/10
Intracytoplasmic proteinaceous droplets !
-respiratory epithelium: in 8/10 in 9/10 ! 1/10 0/10
Mediastinal lymph node !
-macrophage accumulation: (very) slight in 8/10 (very) slight in 8/10 ! 0/10 0/10
------------------------------------------------------------------------------------------------------------------------------



HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable): no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period:
on the average 0.1 - 0.2 mg per lung of male animal groups (not dose-related), 0.05 - 0.21 mg per lung of female groups (dose-related).
Only one male exposed to 30 mg/m3 showed a small amount of silica in the regional lymph node.
90 days after termination of exposure (day 188), no silica could be recovered from any animal.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
1.3 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: Histopathology: based on the slight and fully reversible pulmonary response noted at this exposure level (inflammation reaction) (see details below "Details on results")
Dose descriptor:
LOAEC
Effect level:
5.9 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: (haematology); organ weights (hypertrophy lung); histopathology (collagen increase, sporadic focal fibrosis)
Dose descriptor:
NOEC
Effect level:
< 1.3 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: Histopathology: based on the pulmonary response (inflammation reaction) (see details below "Details on results")

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Please refer to attached document 'Tables and figures' for more information on the results from this study

Findings for quartz-exposed rats:

Clinical signs:None

Body weight:The body weight of quartz-exposed rats was not affected during the exposure period. The rats showed a slightly progressive reduction in weight gain throughout the post exposure period.

Haematology:There was a statistically significant increase in neutrophilic leucocyte counts during the exposure period. The counts increased during the first 13 weeks after the end of the exposures and remained high during the whole post exposure period. Red blood cell counts, haemoglobin content and packed cell volumes had slightly increased in males exposed to quartz by the end of the exposure period. The males continued to show high red blood cell values throughout the observation period. The remaining haematological parameters that were examined did not show differences that could be related to treatment.

Clinical chemistry:From 13 weeks after exposure alanine aminotransferase activities increased in quartz-exposed rats. In males the increase was approximately 50-90% (p<0.01) in comparison with controls. Alkaline phosphatase activity had increased in rats exposed to quartz 52 weeks after the exposure period. The remaining biochemical parameters that were examined did not show differences that could be related to treatment.

Organ weights:At the end of the exposure period both absolute and relative lung weight were statistically significantly increased compared to controls. The increase was greater in males than in females. Lung weight increased progressively during the post-exposure period to levels three or more times higher than those of control animals. The only other organ that showed increased weight was the thymus. Both the absolute and relative thymus weights were significantly increased (by about 140% of control weight in males at day 188). This increase was most pronounced in males and had disappeared 39 weeks after the end of exposure.

Lung collagen contents:At the end of the exposure period, the lung collagen content of quartz exposed rats was slightly higher than in controls, but in the course of the post-exposure period it had increased markedly.

Silicon in the lungs and associated lymph nodes:Silicon levels in the lungs of males were higher 26 weeks after the end of exposure than at 13 weeks after the end of exposure. Males rats exposed to quartz exhibited even higher values at week 39 than week 26 after exposure. This finding was not observed in females, but this could not be explained by the authors.

Pathology:Most of the rats exposed to quartz and killed at the end of the exposure period had swollen and spotted lungs with a spongy consistency and/or irregular surface and enlarged lung-associated lymph nodes. These changes were more pronounced than in the amorphous silica-exposed rats. The gross lesions in the lungs and lung-associated lymph nodes remained present during the whole post-exposure period. Microscopic changes were mainly observed in the lungs. Changes in rats killed at the end of the exposure period comprised slight to severe accumulation of alveolar macrophages, intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes in the alveolar spaces and increased septal cellularity, seen as an increase in the number of type II pneumocytes and macrophages within the alveolar walls. During the post-exposure period no recovery from lung lesions was observed in quartz-exposed rats. Accumulation of intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes were found in all quartz-exposed rats during the post-exposure period. Alveolar broncholization persisted mainly in quartz-exposed animals. Focal interstitial fibrosis, seen as amorphous eosinophilic, collagen-containing thickenings of the septa, was first observed 13 weeks after exposure and became more severe during the post exposure period. Alveolar cholesterol clefts were observed for the first time after 26 weeks of non-exposure. During the remaining recovery period this lesion became more pronounced. Granulomas, seen as aggregates of macrophage-like cells were observed in nearly all rats exposed to quartz at the end of the exposure period. Slight fibrosis was demonstrated in the granulomas in animals of the quartz group. One year after the end of the exposure period, one male rat had a focus of squamous metaplasia in the periphery of the lung. In addition, in one female a small but unequivocal squamous cell carcinoma was found in the lung parenchyma.

Applicant's summary and conclusion