Zeolite, phosphor and titanium containing, crystalline, synthetic, non fibrous

Repeated dose toxicity: inhalation

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:

27 Jul 2001 - 5 Jun 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions from read across substance. MMAD measured once monthly, histopathology for respiratory tract only.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

adopted Sep 2009

Deviations:

yes

Remarks:

MMAD measured once monthly, histopathology for respiratory tract only

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Zeolite, phosphor and titanium containing, crystalline, synthetic, non fibrous

Molecular formula:

M2/nO [TiO4/2]t [SiO4/2]x [AlO4/2]y [PO4/2]z * wH2O, n is the valence of the cation M, predominantly Na and H, and for both is n=1; x and z can range from 0.25 to 0.75, y and t can range from 0.05 to 0.3 and x+y+z+t=1, and w is the number of water molecules typically between 4 and 40. The composition based on the oxides present in the zeolite is typically: Al: 14-27 % K/Na: <5 % Si: 0.1-5 % P: 12-25 % Ti: 0.05-5 %

IUPAC Name:

Zeolite, phosphor and titanium containing, crystalline, synthetic, non fibrous

Details on test material:

- Name of test material (as cited in study report): SAPO 11 calcined powder (experimental product), synthetic non-fibrous zeolite analog, silicoaluminophosphate
- Physical state: powder
- Analytical purity: no data
- Lot/batch No.: 09038-82

Test animals

Species:

rat

Strain:

other: Wistar Crl:(WI)WU

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: approximately 250 g (males), approximately 175 g (females)
- Housing: Two animals per cage in Makrolon cages type III (800 cm²). Cages and softwood ('altromin 3/4') bedding material were changed twice a week or more often when necessary.
- Diet: pellets, altromin 1324 N spec. prepared (Altromin International, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Test substance: 3.2 µm
Titanium dioxide: 1.0 ± 2.06 µm
Quartz: 1.3 ± 2.06 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past, nose-only exposure chamber
- Method of holding animals in test chamber: restrained in acryl tubes with a flexible stopper
- System of generating particulates/aerosols: The aerosol was generated by a high-pressure pneumatic disperser.
- Temperature, humidity, pressure in air chamber: 22 ± 2°C, 40 - 70%,
- Air flow rate: approximately 1 L/min
- Method of particle size determination: cascade impactor

TEST ATMOSPHERE
- Brief description of analytical method used: The aerosol concentrations were measured continuously by light scattering aerosol photometers. The aerosol photometers for the measurement of the aerosol concentration were checked once a week by collecting and subsequently weighing filter samples. The actual aerosol concentrations were calculated using the photometer values, based on weekly gravimetric samples. The average aerosol concentration was in the range of 10% of the target concentration. The aerosol photometer signal was used in the computerized feedback loop to control powder feeding rate. Gravimetrical measurement was performed once per week and the MMAD was determined by cascade impactor once per month.
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The MMAD was determined for each dose once every month. The actual concentrations over the 15-week period, i.e. the actual mean values, were all
within the ± 10% range of the target concentrations.

Duration of treatment / exposure:

90 days and 90 days post-exposure observation period (test group)

Frequency of treatment:

6 h/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.45, 1.9 and 10 mg/m³
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.47 ± 0.19, 1.94 ± 0.37 and 9.88 ± 2.34 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

31 (main group, control groups)

Control animals:

other: yes, air... (see attached file)

Details on study design:

- Rationale for selecting satellite groups: to assess the reversibility of any adverse effects during the recovery period
- Post-exposure recovery period in satellite groups: 90 days

Positive control:

A group of 31 rats/sex were exposed to 7.2 mg/m³ quartz as the positive control. The animals were treated according the same protocol as the treatment groups.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, the food consumption of 10 animals/group was recorded weekly.

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: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Yes, overnight for 16 h
- How many animals: 10 animals/group
- Parameters examined: red blood cells (RBC), total white blood cells (WBC), hemoglobin (HB), differential white cell count (% and absolute), hematocrit (HCT), platelets (PTL), mean cell volume (MCV), mean hemoglobin/erythrocyte (MCH), mean hemoglobin concentration/erythrocyte (MCHC), prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: Yes, overnight for 16 h
- How many animals: 10 animals/group
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), urea, creatinine (CREA), total protein (TP), albumin (ALB), globulin (GLB), ALB/GLB, glucose (GLUC), cholesterol (CHOL), sodium (Na), calcium (Ca), potassium (K)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Lung burden measurements:
The similarity of the lung burden in the control, the zeolite analog groups, the quartz group and the positive control group was assessed. The right lung lobes were freeze-dried and subjected to low-temperature ashing. The remaining ash was analyzed for aluminum in the zeolite analog, silicon in the DQ12 groups and for titanium in the titanium dioxide group using atom absorption spectroscopy (AAS) after wet digestion. Background levels of aluminum, silicon and titanium were determined in lungs of the control group. Potential leaching of aluminum from the zeolite analog crystal was considered to be negligible. The lung-associated lymph nodes (LALN) of the rat lungs used for retention measurement were examined histopathologically. To make this organ available for retention measurements, the LALN from lungs used for BAL were prepared and data of pooled group samples were determined. 60 lungs (5 animals/group and time point) and 12 pooled LALN samples were processed each.

Bronchoalveolar lavage (BAL):
Performed in 5 rats per dose and time point (after end of exposure and after 90 days of post-observation). Leukocyte concentration of the lavagate was determined using a counting chamber, and two cytoslides were prepared with a cytocentrifuge for differential cell count (macrophages, neutrophils, eosinophils, lymphocytes) and analysis of epithelial cells. Another aliquot was used for a viability assay of lavaged cells (trypan blue exclusion test). After centrifugation of the lavage fluid, biochemical indicators relevant for diagnosis of lung damage were determined in the supernatant (lactic dehydrogenase, glucuronidase, total protein).

BrdU-proliferation assay:
Formalin-fixed tissues of the terminal bronchioles and lung parenchymal cells were examined for cell proliferation using the sensitive S-phase response method (10 animals/dose group and time point). Proliferating cells were labeled by 5-bromo-2'-deoxyuridine (BrdU; 20 mg/mL in phosphate-buffered saline) which was administered to the animals by a minipump 6 days prior to sacrifice. The lung section slides were prepared according to histological routine procedures and stained immunohistochemically following denaturation of the DNA (antibody technique). The slides were evaluated by analyzing an appropriate number of airway cells and cells of the proximal regions of the pulmonary parenchyma per rat. Four fields of parenchyma (top, below, right and left to the terminal bronchiolus at a distance of 1 alveolus between the counting frame and the bronchiolar wall) per evaluated bronchiolus were analyzed. A minimum of 2000 cells were counted (400fold magnification) per animal. The Unit Length Labeling Index of at least 4 terminal bronchioli (longitudinally sectioned) was determined using light microscopy (400fold magnification) and an image analysis system. The number of BrdU-positive cells was recorded on a length of approximately 500 mm of bronchiolar epithelium, counting from the bronchiolo-alveolar transition towards the proximal terminal bronchiole (both sides of the terminal bronchiolus were evaluated).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were subjected to a complete necropsy, which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The rats used for bronchoalveolar lavage (BAL) were anesthetized with an overdose pentobarbital sodium (Narcoren®) and killed by cutting the vena cava caudalis. Rats for histopathological investigations were sacrificed with an overdose of carbon dioxide. The abdominal cavity was opened and the diaphragm was cut carefully allowing the lungs to collapse. Heart, esophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN) were removed from the lung convolution. For lung retention and histopathology, the lung and the lower half of the trachea were weighed. After putting a tourniquet on the main bronchus to the right lung lobes, these lobes were cut off at the lung tissue border and used for lung retention measurement. Prior to the start of the analysis, the right lung lobes and the upper half trachea were weighed and stored in plastic tubes at -25 ± 5°C. The left lung was inflated under a pressure of about 20 cm water with formalin, fixed by immersion for a minimum of two hours, and used for histopathology. Thereafter, the weight of the lower part of the trachea was recorded and the weight of the left lobe was calculated as difference between total lung including the lower part of the trachea and the sum of the right lung lobes and lower part of the trachea.
From the animals used for histopathology the following organs were weighed and wet weights were recorded: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, and heart.

HISTOPATHOLOGY: Yes. In 10 animals from the control and high-dose groups, per time point (after end of exposure and after 90 days of post-observation), and in animals that died or were killed during the study period, full histopathology was performed on the respiratory tract and specified organs and tissues. Furthermore, histopathology of the lung lobes, including bronchi and the lung-associated lymph nodes was performed. Lungs were fixed in buffered formalin (10%), embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E). A special stain (Masson trichrome) was applied for diagnosis of fibrotic changes. These histopathological examinations were done on the lower half of the left lobe.

Statistics:

Differences between groups were considered to be statistically significant at p < 0.05. Data were analyzed using analysis of 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 using Tukey's test. Body weight and food consumption data which have been recorded by the DATATOX system (Tukey's test not available) were evaluated using the L.S.D. test. The statistical evaluation of the histopathological findings was done with the two-tailed Fisher test by the P.L.A.C.E.S. system. In the Fisher test the treated groups were compared to the clean air control group.

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):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

10 mg/m³: increased mean absolute and mean relative segmented neutrophiles (non-adverse)

Clinical biochemistry findings:

no effects observed

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):

10 mg/m³: increased relative wet lung weight, females

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

0.45 mg/m³: pulmonary inflammation, bronchio-alveolar hyperplasia, alveolar accumulation of particle-laden macrophages, accumulation of particle-laden macrophages in lung-associated lymph nodes

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
1/31 males in the mid-dose zeolite group, 2/31 males and 1/31 females in the titanium dioxide group, and 2/31 males and 1/31 females in the quartz group, died or were killed in extremis during the study period. None of the deaths were toxicologically relevant. No treatment-related clinical signs were observed during the study period.

BODY WEIGHT AND WEIGHT GAIN
The body weight gain during the study period was within the normal range known for rats of this sex and strain.

FOOD CONSUMPTION
Significant differences in food consumption were noted in all zeolite treatment groups, quartz group and titanium dioxide group, compared with the control, at one or more measuring points during the study period. However, as the changes were not consistent regarding increase or decrease, did not show a trend over time, and were not dose-dependent, these are considered to be incidental changes.

HAEMATOLOGY
In the high-dose zeolite females and the quartz group, a significant increase in mean absolute and mean relative segmented neutrophiles was reported (see Table 1 under 'Any other information on results incl tables'). This effect is considered to be related to the pulmonary inflammation.

CLINICAL CHEMISTRY
No treatment-related effects were observed in the zeolite-treated groups. In females in the titanium dioxide group, a slight (< 10%), though significant, decrease in calcium levels was noted.

ORGAN WEIGHTS
The relative wet lung weight of high-dose zeolite females (main group) was significantly increased, compared with control. The quartz main group and recovery group showed the expected increase in relative and absolute wet lung weight, compared with control.

GROSS PATHOLOGY
See histopathology below.

HISTOPATHOLOGY: NON-NEOPLASTIC
8 day post-exposure examination:
Lungs:
No lesions were observed in the control male lungs (see Table 4 under 'Any other information on results incl tables' and Table 5 under 'Attached background material'). Single females of this group showed focal very slight (minimal) to slight interstitial fibrosis, pleural fibrosis or alveolar histiocytosis. Some of these lesions were associated with spontaneous foreign-body granulomas which were seen in one female of this group. Nearly all rats of the particle inhalation treatment groups had dose-dependent multifocal, very slight (minimal) to moderate alveolar accumulations of particle-laden macrophages (0.45 mg/m³ zeolite: 8/10 males, 10/10 females; 1.9 and 10 mg/m³ zeolite: 10/10 males and females per group; quartz and titanium dioxide: 10/10 males, 11/11 females per group). One male in the quartz group showed severe alveolar accumulation of particle-laden macrophages. In all exposure groups, the alveolar macrophages were preferentially located in the alveolar duct region and appeared cytologically intact in the zeolite and titanium dioxide groups. In the quartz group, they were mainly foamy and degenerated, as also reflected by alveolar lipoproteinosis. Mainly moderate and multifocal to diffuse alveolar lipoproteinosis was observed in all quartz group rats (10/10 males, 11/11 females), where the lipoproteinaceous intra-alveolar material appeared to be mainly derived from decaying alveolar macrophages. Interstitial accumulations of particle-laden macrophages were observed at increasing incidences and with very slight (minimal) to slight severity in the zeolite groups: 0.45 mg/m³: 1/10 males, 3/10 females; 1.9 mg/m³: 8/10 males, 9/10 females; 10 mg/m³:
10/10 males and females each). In the quartz group, 10/10 males and 11/11 females showed mainly slight interstitial accumulations of particle-laden macrophages. Focal or multifocal interstitial (predominantly perivascular) mononuclear cell infiltration was seen dose-dependently with mainly very slight (minimal) to slight severity in the zeolite groups (0.45 mg/m³: 0/10 males, 4/10 females; 1.9 mg/m³: 8/10 males, 10/10 females; 10 mg/m³: 10/10 males and 10/10 females) and slight to moderate severity in all quartz-exposed animals (10/10 males, 11/11 females). In the titanium dioxide group, only 1/11 females showed a very slight (minimal) interstitial mononuclear cell infiltration. The inflammatory activity in the lungs was evident by the degree of granulocytic infiltration of alveoli. This change was not observed in the titanium dioxide group but occurred with mainly moderate severity in all rats of the quartz group (10/10 males, 11/11 females). Granulocytic infiltration was seen dose-dependently in the low- and mid-dose zeolite groups (0.45 mg/m³: 3/10 males, 6/10 females; 1.9 mg/m³: 10/10 males and females each) with a very slight (minimal) severity. In the 10 mg/m³ zeolite group, 8/10 males and females each showed a very slight (minimal) alveolar granulocytic infiltration, and 2/10 males and females each had slight alveolar granulocytic infiltration. Dose-dependent multifocal bronchiolo-alveolar hyperplasia, mainly of the bronchiolar type was also noted; with very slight (minimal) severity (1/10 males of the 10 mg/m³ dose group: slight severity in the zeolite groups (0.45 mg/m³: 1/10 males, 0/10 females; 1.9 mg/m³: 3/10 males and females each; 10 mg/m³: 6/10 males and females each). All rats of the quartz group (10/10 males, 11/11 females) had multifocal, mainly slight bronchiolo-alveolar hyperplasia, while in the titanium dioxide group, only one male showed this change. The hyperplastic epithelium often consisted of only few cells and usually developed in association with inflammatory and fibrotic foci. In the quartz group, however, many of the hyperplasias were of the mixed type and consisted of a mixture of hyperplastic bronchiolar cells and alveolar type-II cells. Interstitial fibrosis which mainly affected the bronchiolo-alveolar junctions was not detected in animals of the 0.45 mg/m³ zeolite group or in males of the titanium dioxide group. Two out of 10 males and 1/10 females of the 1.9 mg/m³ zeolite group showed a very slight (minimal) multifocal interstitial fibrosis, as demonstrated with the Masson trichrome stain. In the 10 mg/m³ zeolite group, all males (9/10 very slight, 1/10 slight) and 9/10 females (all very slight) showed this change, while all rats treated with quartz were affected by multifocal interstitial fibrosis of slight severity (10/10 males, 11/11 females).

Lung-associated lymph nodes (LALN):
The LALN of nearly all rats of the particle treatment groups showed an accumulation of particle-laden macrophages. In the zeolite groups, these were dose-dependent (incidences between 6/10 and 10/10 per sex and group) and predominantly very slight (minimal) in the 0.45 mg/m³, slight in the 1.9 mg/m³ and moderate in the 10 mg/m³ dose group, respectively. Mainly severe accumulation of particle-laden macrophages was diagnosed in the quartz treated rats (10/10 males, 11/11 females), whereas 8/10 males and 11/11 females of the titanium dioxide group had mainly very slight (minimal) foci of macrophages. Associated with the accumulation of particle-laden macrophages were slight to moderate reactive lymphoid hyperplasias, which were seen at low incidences and with slight severity (range 1/10 to 2/11 animals per group) in both the low-dose zeolite and the titanium dioxide group. In the med- and high-dose zeolite groups and in the quartz group, between 5/10 and 11/11 rats per group had slight to moderate lymphoid hyperplasia. In the quartz group, 4/10 males and 6/11 females exhibited additional slight to severe foci of granulomatous inflammation with necrosis and infiltration of granulocytes and occasional multinucleated giant cells.
Nasal cavity, larynx, trachea:
Slight focal mucous (goblet) cell hyperplasia was observed in up to 3/10 males and 2/10 females per group of the high-dose zeolite group, the quartz and the titanium dioxide groups. In addition, very slight multifocal eosinophilic cytoplasmic inclusions of respiratory and olfactory epithelial cells were noted in up to 2/10 rats of these groups. Although the difference to the control group was not significant, these findings may be related to exposure.

No toxicologically relevant changes were noted in other organs or tissues.

Results from the 3-month recovery group
All rats in the particle inhalation treatment groups had dose-dependent multifocal, very slight (minimal) to moderate alveolar accumulations of particle-laden macrophages (zeolite, quartz and titanium dioxide: 10/10 males and females each group). Two out of ten males and females each of the quartz group showed severe alveolar accumulation of particle-laden macrophages. As in the 1-day postexposure group, the macrophages were preferentially located in the alveolar duct region and appeared cytologically intact in the zeolite and titanium dioxide groups, however, they were mainly foamy and degenerating in the quartz group. In addition, moderate to severe, multifocal or diffuse alveolar lipoproteinosis was also observed in rats of the quartz group (10/10 males and females each), where the lipoproteinaceous intra-alveolar material appeared to be mainly derived from decaying alveolar macrophages. Interstitial accumulations of particle-laden macrophages were observed at increasing incidences and with very slight (minimal) to slight severity in the zeolite groups (0.45 mg/m³: 1/10 males, 3/10 females; 1.9 mg/m³: 8/10 males, 9/10 females; 10 mg/m³: 10/10 males and females each). In the quartz group, 10/10 males and 11/11 females showed mainly slight interstitial accumulations of particle-laden macrophages, while no rats in the titanium dioxide group showed this finding. In comparison to the 1-day postexposure group, no substantial difference was observed with respect to the incidences of alveolar and interstitial macrophage accumulation at the 3-month postexposure interval, although the severity of these findings in the zeolite groups, especially in the females of the high dose group, had decreased. Focal or multifocal interstitial (predominantly perivascular) mononuclear cell infiltration was seen dose-dependently with mainly very slight (minimal) to slight severity in the zeolite groups (0.45 mg/m³: 2/10 males, 1/10 females; 1.9 mg/m³: 6/10 males, 3/10 females; 10 mg/m³: 10/10 males, 5/10 females) and slight to moderate severity in all quartz-treated animals (10/10 males and females each. In comparison to the 1-day postexposure period, the incidences and severities of interstitial mononuclear cell infiltration were markedly lower in the zeolite and titanium dioxide groups 3-month postexposure. As with other findings, no such decrease was observed for the quartz group. Alveolar granulocytic infiltration was not observed in the titanium dioxide group; however, it was diagnosed with slight to moderate severity in all rats of the quartz group (10/10 males and females each). In the zeolite groups, this lesion occurred dose-dependently (0.45 mg/m³: 2/10 males, 3/10 females; 1.9 mg/m³: 10/10 males, 8/10 females; 10 mg/m³: 10/10 males and females each) with very slight (minimal) severity (except for one male of the high dose zeolite group with slight alveolar granulocytic infiltration). With respect to this finding, there was no substantial difference between the two postexposure groups. The same refers to bronchiolo-alveolar hyperplasia and interstitial fibrosis, but only in the zeolite and titanium dioxide exposure groups and not in the quartz group. Dose-dependent multifocal bronchiolo-alveolar hyperplasia occurred with very slight (minimal) severity in the zeolite groups (0.45 mg/m³: 3/10 males, 1/10 females; 1.9 mg/m³: 5/10 males, 0/10 females; 10 mg/m³: 5/10 males, 2/10 females). All rats of the quartz group (10/10 males and females each) had multifocal, mainly slight bronchiolo-alveolar hyperplasia, while in the titanium dioxide group, only one male showed this change. In the zeolite and titanium dioxide groups, hyperplasias of the bronchiolar type (alveolar bronchiolization) were observed, whereas the vast majority of hyperplasias in the quartz group were of the mixed type. In contrast to the bronchiolar type, the mixed type of bronchiolo-alveolar hyperplasia has to be considered as a potentially pre-neoplastic lesion. Interstitial fibrosis was not detected in the 0.45 mg/m³ zeolite group or in the titanium dioxide group. Four out of 10 males and 1/10 females of the 1.9 mg/m³ zeolite group showed very slight (minimal) multifocal interstitial fibrosis, while in the 10 mg/m³ zeolite group, 9/10 males and 6/10 females had very slight (minimal) interstitial fibrosis. In the quartz group, slight interstitial fibrosis was diagnosed in 9/10 males and females each, while 1/10 rats of each sex had moderate interstitial fibrosis, indicating an increase of collagen deposition when compared to the 8-day postexposure group. In the quartz group, pronounced interstitial fibrosis especially developed around cholesterol granulomas. Multifocal cholesterol granulomas with slight to moderate severity were seen in 10/10 males and 7/10 females of the quartz group. In addition, 1/10 females of the titanium dioxide group had a focal cholesterol granuloma with slight severity. This change was not observed in any rat of the zeolite groups.

Lung-associated lymph nodes (LALN):
The LALN of nearly all rats of the particle treatment groups showed accumulations of particle-laden macrophages. In the zeolite groups, these were dose-dependent (incidences between 8/10 and 10/10 per sex and group) and were predominantly slight in the 0.45 mg/m³, slight to moderate in the 1.9 mg/m³ and moderate in the 10 mg/m³ dose group, respectively. Severe accumulations of particle-laden macrophages were diagnosed in quartz-exposed rats (10/10 males and females each), whereas 9/10 males and females each of the titanium dioxide group had mainly very slight (minimal) foci of macrophages. Slight to moderate reactive lymphoid hyperplasia was associated with the macrophage accumulations, which in the low-dose zeolite and titanium dioxide groups had low incidences and appeared with mainly slight severity (range 2/10 to 5/10 animals per group). In the medium and high-dose zeolite groups and in the quartz group, between 5/10 and 10/10 rats per group had lymphoid hyperplasia, which in the quartz group was mainly severe and in the zeolite groups slight to moderate. Only in the quartz group, 4/10 males and 1/10 females exhibited additional slight to moderate foci of granulomatous inflammation with necrosis and infiltration of granulocytes and occasional multinucleated giant cells.

Nasal cavity, larynx, trachea:
Slight focal mucous (goblet) cell hyperplasia of the nasal cavity was observed in up to 1/10 males and females each per group with a predominance in males. In addition, slight focal mucosal inflammatory cell infiltration, slight focal mucosal mineralization and slight cystic
dilatation of submucosal glands affected single males of the high dose zeolite analog and quartz groups.

No toxicologically relevant changes were noted in other organs or tissues.

OTHER FINDINGS

Lung burden measurements:
The analysis of lung retention including lung associated lymph nodes demonstrated similar initial lung burdens in the high-dose zeolite group compared with the reference substances quartz and titanium dioxide. In comparison with the titanium dioxide group, only very slight retardation of lung clearance was observed in the high dose zeolite group (see Table 2 under 'Any other information on results incl tables').

BAL:
One day after the exposure ended, the BAL analysis showed levels of polymorphonuclear neutrophils (PMN) of 9%, 40% and 49% (sexes pooled) in the zeolite low-, mid- and high dose group, respectively (see Table 3 under 'Any other information on results incl tables'). In the quartz and titanium dioxide group the values were 51% and 0.5%. After a 3-month recovery period the corresponding data were 10%, 35% and 56% in the zeolite low-, mid- and high dose groups and 58% and 1.6% in the quartz and titanium dioxide groups. The zeolite mid- and high-dose induced a moderate to severe lung inflammation in lungs persisting over three months. However, the response was significantly weaker than that of the quartz group (for zeolite mid- and high-dose after one day/zeolite mid-dose after three months). The absolute leukocyte numbers were 465,000, 845,000 and 2,000,000 cells (sexes pooled) in the zeolite low-, mid- and high dose group 1 day after termination of exposure. In the quartz and titanium dioxide group the values were 31,400,000 and 640,000 cells. The leukocyte numbers of all zeolite groups and the titanium dioxide groups were close to or in the range of the control group (475,000 cells).
The trypan blue exclusion assay for cell viability did not show treatment-related effects.
The epithelial cell number was significantly decreased in the quartz group (1 day and 3 months after exposure ended). There were no significant differences between the zeolite treatment groups and control, however, there was a clear decrease in the zeolite high-dose group, indicating partial necrosis of the bronchiolar lining epithelium.
Mild dose-related increases in mean values were observed for lactic dehydrogenase, β-glucuronidase, and total protein levels in males and females. However, significant increases in total protein was only observed in males in the mid- and high-dose zeolite group. In the quartz group, significant increases in lactic dehydrogenase, β-glucuronidase, and total protein levels were observed.

BrdU-proliferation assay:
In both sexes, the 6-bromo-deoxyuridine (BrdU) proliferation assay on lung parenchyma resulted in a dose-dependent increase of labeling indices in the zeolite analog groups (approx. 2.8, 4.8 and 6.5% vs. approx. 2.5% in controls 8 days after termination of exposure; all pooled data). In the female mid- and high-dose groups this increase was statistically significant (see Table 6 under 'Any other information on results incl tables'). In the quartz group the increase was approx. 14% and in the titanium oxide group about 3%. After 3 months of recovery only the female zeolite high-dose group remained significantly increased when compared to the controls. At all time points, the quartz groups differed significantly from the zeolite mid- and high-dose groups. In terminal bronchiolar epithelium, the zeolite groups did not show clear significant increases. Here, the quartz groups were significantly increased when compared to the controls (with the exception of day 91, females).

Effect levels

Dose descriptor:

NOAEC

Effect level:

< 0.45 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Pulmonary inflammation (irreversible), bronchio-alveolar hyperplasia, alveolar accumulation of particle-laden macrophages, accumulation of particle-laden macrophages in lung-associated lymph nodes. Equivalent to 0.00045 mg/L.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Selected hematology results (numbers given in bold are statistically significant compared with air control)

Parameter	Air control	Zeolites Low dose	Zeolites Medium dose	Zeolites High dose	Quartz	Titanium dioxide
Hematology
Study days post exposure	1	1	1	1	1	1
- segmented neutrophils (absolute values)	1.31 g/L (m) 0.56 g/L (f)	1.05 g/L (m) 0.90 g/L (f)	1.14 g/L (m) 0.80 g/L (f)	1.31 g/L (m) 1.05 g/L (f)	1.85 g/L (m) 1.38 g/L (f)	1.07 g/L (m) 0.60 g/L (f)
- segmented neutrophils (relative values)	10% (m) 8% (f)	10% (m) 11% (f)	11% (m) 11% (f)	12% (m) 13% (f)	16% (m) 17% (f)	10% (m) 9% (f)

 

Table 2: Lung retention results (numbers given in bold are statistically significant compared with air control)

Parameter	Air control		Zeolites Low dose		Zeolites Medium dose		Zeolites High dose		Quartz		Titanium dioxide
Lung retention/lung clearance
Study days post exposure	8	91	8	91	8	91	8	91	8	91	8	91
Test substance retained in lungs (in µg)	5 5	4 (m) 4 (f)	123 91	36 (m) 27 (f)	438 276	132 (m) 86 (f)	1115 933	519 (m) 409 (m)	1081 993	908 (m) 1007 (f)	1193 1084	573 (m) 332 (f)
Test substance retained in lymph nodes (in µg)	0.1 0.0	0.2 (m) 0.2 (f)	1.5 0.8	2.0 (m) 1.3 (f)	9.3 7.0	29.0 (m) 20.7 (f)	95.2 42.0	39.3 (m) 33.2 (f)	94.4 104.2	261.0 (m) 182.4 (f)	0.6 0.6	2.0 (m) 1.4 (f)
Percentage of substance retention¹	n.a.	n.a.	10.2 9.4	3.2 (m) 2.9 (f)	37.0 29.0	13.3 (m) 10.9 (f)	100.0 100.0	46.1 (m) 45.3 (f)	97.1 112.5	96.6 (m) 122.0 (f)	98.6 111.3	47.5 (m) 34.2 (f)

1.    Based on test substance amount on study day 8 of the high dose group

 

Table 3: Broncho-alveolar lavage results (numbers given in bold are statistically significant compared with air control)

Parameter	Air control		Zeolites Low dose		Zeolites Medium dose		Zeolites High dose		Quartz		Titanium dioxide
Broncho-alveolar lavage
Study days of post exposure	1	91	1	91	1	91	1	91	1	91	1	91
LDH (U/l)	30 36	34 (m) 37 (f)	51 50	42 (m) 49 (f)	73 72	51 (m) 69 (f)	108 133	95 (m) 104 (f)	693 846	665(m) 751(f(	37 59	35 (m) 36 (f)
ß-GLU (U/L)	0.2 0.4	0.1 (m) 0.3 (f)	0.3 0.3	0.2 (m) 0.3 (f)	0.4 0.4	0.1 (m) 0.5 (f)	0.7 0.8	0.4 (m) 0.6 (f)	15.0 30.0	12.6 (m) 16.4 (f)	0.3 0.5	0.2 (m) 0.3 (f)
Protein (mg/L)	97 86	100 (m) 116 (f)	142 126	115 (m) 116 (f)	169 145	148 (m) 162 (f)	261 220	201 (m) 220 (f)	1087 1264	1027 (m) 1201 (f)	100 115	96 (m) 107 (f)
Leukocyte number (x 10^5)	5.6 3.9	6.2 (m) 3.9 (f)	5.3 3.9	5.0 (m) 3.5 (f)	9.5 7.4	5.8 (m) 4.1 (f)	21.4 19.2	8.4 (m) 5.7 (f)	300.6 327.4	132.6 (m) 73.2 (f)	8.0 4.8	5.4 (m) 3.8 (f)
Neutrophils (%)	0.3 0.3	1.0 (m) 1.4 (f)	6.3 12.6	10.2 (m) 10.1 (f)	35.9 43.0	37.3 (m) 31.7 (f)	40.6 57.1	59.1 (m) 53.2 (f)	55.5 45.9	64.9 (m) 50.3 (f)	0.3 0.7	1.0 (m) 2.2 (f)
Macrophages (%)	99.5 99.4	96.0 (m) 97.4 (f)	85.6 85.5	84.9 (m) 84.3 (f)	61.1 54.0	48.8 (m) 55.0 (f)	52.0 40.8	31.8 (m) 34.3 (f)	43.3 52.2	29.5 (m) 44.2 (f)	98.9 98.9	99.0 (m) 97.0 (f)
Lymphocytes (%)	0.3 0.3	1.3 (m) 1.3 (f)	3.2 1.8	4.9 (m) 5.6 (f)	3.0 3.1	13.9 (m) 13.3 (f)	7.4 2.1	9.0 (m) 12.5 (m)	1.2 1.9	5.7 (m) 5.6 (f)	0.9 2.9	5.2 (m) 4.9 (f)
Epithelial cells (No./200 cells)	9.8 10.4	17.8 (m) 30.3 (f)	9.8 6.2	15.8 (m) 20.5 (f)	4.8 8.8	13.8 (m) 7.8 (f)	3.2 1.6	11.2 (m) 7.2 (f)	0.8 0.4	1.4 (m) 1.4 (f)	6.2 17.0	31.8 (m) 13.8 (f)

 

Table 4: Histopathology results (numbers given in bold are statistically significant compared with air control)

Parameter

Air control

Zeolites Low dose

Zeolites Medium dose

Zeolites High dose

Quartz

Titanium dioxide

Histopathology - lung (number of animals (m/f))

Study days post exposure

8

91

8

91

8

91

8

91

8

91

8

91

No. animals examined

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/11

10/10

10/11

10/10

Alveolar accumulation of macrophages Minimal Slight Moderate Severe

0/0     0/0 0/0 0/0 0/0

0/0     0/0 0/0 0/0 0/0

8/10     8/9 0/1 0/0 0/0

10/10     10/10 0/0 0/0 0/0

10/10     9/8 1/2 0/0 0/0

10/10     2/6 8/4 0/0 0/0

10/10     3/4 7/6 0/0 0/0

10/10     2/7 8/3 0/0 0/0

10/11     0/0 1/0 8/11 1/0

10/10     0/0 0/0 8/8 2/2

10/11     10/11 0/0 0/0 0/0

10/10     10/9 0/1 0/0 0/0

Interstitial accumulation of macrophages Minimal Slight Moderate Severe

0/0     0/0 0/0 0/0 0/0

0/0     0/0 0/0 0/0 0/0

1/3     1/3 0/0 0/0 0/0

4/2     4/2 0/0 0/0 0/0

8/9     8/9 0/0 0/0 0/0

10/10     9/10 1/0 0/0 0/0

10/10     8/7 2/3 0/0 0/0

10/10     7/7 3/3 0/0 0/0

10/11     0/0 9/11 1/0 0/0

10/10     0/0 8/0 2/8 0/2

0/0     0/0 0/0 0/0 0/0

2/1     2/1 0/0 0/0 0/0

Interstitial mononuclear cell infiltration Minimal Slight Moderate Severe

0/0     0/0 0/0 0/0 0/0

0/0     0/0 0/0 0/0 0/0

0/4     0/4 0/0 0/0 0/0

2/1     2/0 0/1 0/0 0/0

8/6     4/2 4/4 0/0 0/0

6/3     4/3 2/0 0/0 0/0

10/10     2/2 7/8 1/0 0/0

10/5     5/4 5/1 0/0 0/0

10/11     0/0 8/9 2/2 0/0

10/10     0/0 6/10 4/0 0/0

0/1     0/1 0/0 0/0 0/0

0/0     0/0 0/0 0/0 0/0

Alveolar granulocytic infiltration Minimal Slight Moderate Severe

0/0     0/0 0/0 0/0 0/0

0/0     0/0 0/0 0/0 0/0

3/6     3/6 0/0 0/0 0/0

2/3     2/3 0/0 0/0 0/0

10/10     10/10 0/0 0/0 0/0

10/10     10/10 0/0 0/0 0/0

10/10     7/8 3/2 0/0 0/0

10/10     9/10 1/0 0/0 0/0

10/11     0/0 8/9 2/2 0/0

10/10     0/0 1/6 9/4 0/0

0/0     0/0 0/0 0/0 0/0

0/0     0/0 0/0 0/0 0/0

Broncho-alveolar hyperplasia Minimal Slight Moderate Severe

0/1   0/1 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

1/0   1/0 0/0 0/0 0/0

3/1   3/1 0/0 0/0 0/0

3/3   3/3 0/0 0/0 0/0

5/0   5/0 0/0 0/0 0/0

6/6   5/6 1/0 0/0 0/0

5/6   5/1 0/1 0/0 0/0

10/11   0/0 3/1 7/10 0/0

10/10   0/0 9/9 1/1 0/0

1/0   0/0 1/0 0/0 0/0

1/0   1/0 0/0 0/0 0/0

Interstitial fibrosis  Minimal Slight Moderate Severe

0/1   0/1 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

2/1   2/1 0/0 0/0 0/0

4/1   4/1 0/0 0/0 0/0

10/9   9/9 1/0 0/0 0/0

9/9   9/9 0/0 0/0 0/0

10/11   0/0 10/11 0/0 0/0

10/10   0/0 9/8 1/2 0/0

0/1   0/1 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

Alveolar lipoproteinosis Minimal Slight Moderate Severe

0/0   0/0 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

10/11   0/0 1/0 7/8 2/3

10/10   0/0 0/0 6/1 4/9

0/0   0/0 0/0 0/0 0/0

0/0   0/0 0/0 0/0 0/0

Table 6: Lung proliferation results (numbers given in bold are statistically significant compared with air control)

Parameter	Air control		Zeolites Low dose		Zeolites Medium dose		Zeolites High dose		Quartz		Titanium dioxide
Lung proliferation
Study days post exposure	8	91	8	91	8	91	8	91	8	91	8	91
BrdU-Proliferation Assay (% positive cells)	2.10 2.93	1.94 (m) 3.13 (f)	2.21 3.42	2.20 (m) 3.07 (f)	3.67 5.91	2.33 (m) 3.44 (f)	5.57 7.50	3.60 (m) 6.06 (f)	13.62 14.80	10.73 (m) 11.71 (f)	2.17 3.66	1.70 (m) 2.62 (f)

 

Applicant's summary and conclusion

Conclusions:

Based on the irreversible pulmonary inflammation observed at 0.45 mg/m³, the substance is classified STOT RE Cat. 2, H373.