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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study with acceptable restrictions (publication with deficiencies in documentation)

Data source

Reference
Reference Type:
publication
Title:
Pulmonary response to toner upon chronic inhalation exposure in rats
Author:
Muhle, H. et al.
Year:
1991
Bibliographic source:
Fundam Appl Toxicol. 17(2):280-99.

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
limited documentation
GLP compliance:
not specified
Remarks:
GLP study according to the authors. No further details on testing laboratory given.
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Quartz (SiO2)
EC Number:
238-878-4
EC Name:
Quartz (SiO2)
Cas Number:
14808-60-7
Molecular formula:
O2Si
IUPAC Name:
dioxosilane
Details on test material:
- Name of test material (as cited in study report): DQ-12 (crystalline silicon dioxide)
- Analytical purity: no data
- Crystallinity: 87% crystallinity as alpha-quartz
- Source: Bergbauforschung, D-4300, Essen. Germany

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga GmbH
- Age at study initiation: 8 weeks
- Housing: Rats were individually housed in metal wire mesh cages (18.7 X 21 X 15 cm) in horizontal air flow chambers throughout the study.
- Acclimation period: 4 weeks


Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: 1.4 µm/ 1.8 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure facility and general procedures have been described (Muhle et al., 1990a, Inhal. Toxicol. 2:341-359).
- Method of holding animals in test chamber: Whole-body exposure chambers were used.
- Source and rate of air: pressurized air
- System of generating particulates/aerosols: A dry aerosol dispersion technique was used. The aerosol generator consisted of a commercially available feeding system connected to a two-stage pressurized air ejector. Details of the aerosol generator feeder system as well as the concentration monitoring equipment and the 12-m3 horizontal flow inhalation chambers have been published (Koch et al., 1986, J Aerosol Sci. 3:499-504; Heinrich et al., 1985, Safety Evaluation and Regulation of Chemicals (F. Homburger, Ed.), pp. 239-250). To maximize mixing and uniform distribution of the aerosol, the chamber inlets were equipped with diffusers and perforated plates.
- Temperature, humidity in air chamber: 23.5 ± 1 °C and 40-60% relative humidity
- Air flow rate: 3.8 m3/min
- Method of particle size determination: The particle size distribution in each chamber was measured 13 times during the study using a Bemer impactor. The measured values at this position were about 10% higher compared to the mean (27 sampling positions) for the whole chamber. This difference was taken into consideration during calibration. The gravimetric values were used for periodic recalibration of the aerosol photometer.


TEST ATMOSPHERE
- Brief description of analytical method used: At the inlet side of the chamber photometric determination of the aerosol concentration was done and gravimetric samples were collected.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No details given
Duration of treatment / exposure:
6 h/day
Frequency of treatment:
5 days/week for 24 months
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1.0 +/- 0.1 mg/m³
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0.74 +/- 0.04 mg/m³
Basis:
other: Respirable aerosol concentration
No. of animals per sex per dose:
144, thereof a total of 22 animals were sacrificed at intervals.
Control animals:
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Microbiological status was investigated on arrival, 2 and 4 weeks subsequently, and thereafter at 6-month intervals. Clinical laboratory tests at 6, 12, 18, and 25.5 months.


BODY WEIGHT: Yes



FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


HAEMATOLOGY: Yes


CLINICAL CHEMISTRY: Yes


URINALYSIS: Yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Animal sacrifice and tissue processing Rats sacrificed during and after scheduled exposure were anesthetized with sodium pentobarbital and killed by cutting the abdominal aorta. The abdominal cavity was opened and the diaphragm was cut allowing the lungs to collapse. The specified organs (brain, liver, kidneys, adrenals, and gonads) were weighed. Tissues and organs were fixed in 10% neutral buffered formalin and processed for routine histology. Bones were decalcified with Decal. The larynx, trachea, esophagus, thymus, heart, and lungs were removed en masse. The larynx and upper part of the trachea were separated and placed in formalin. For lungs scheduled for histopathology, the lower part of the trachea was cannulated and the lungs were fixed under inflation at 20 cm water pressure for 24 hr with Ito-Karnovsky fixative. Following fixation, the trachea was tied off; the lungs were placed for 8-24 hr in 70% ethanol and stored in 10% neutral formalin.
HISTOPATHOLOGY: Yes. Complete histopathological examination of organs, tissues, and gross lesions of animals of the serial sacrifices and of all animals of the "basic study" from the control and high-dose groups was performed. Only the respiratory system (nasal and paranasal cavities, larynxpharynx, trachea, lungs, lung-associated lymph nodes) was examined in the other 400 animals constituting the "basic study."
Statistics:
Parametric data were examined by analysis of variance (ANOVA) followed by Dunnett test to compare various treatment groups with controls. Survival
data were analyzed by the Kaplan-Meier method using the lifetest program of SAS (SAS Institute, 1985, SAS User's Guide. Statistics, Version 5 ed. SAS Institute Inc.. Cary, NC). For necropsy and tumor occurrence data, simple tests for homogeneity of contingency tables (using chi-square statistics or Fisher's exact method) were used.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
Inhalation of the various test materials did not cause overt signs of toxicity. The clinical appearance of the rats was found to be within normal limits in all groups.
The survival data for various groups are shown in Fig. 1 (refer to attached background material). The mean survival in the basic study (100 animals, 50 of each gender) was 35% for the SiO2 group. The three most frequent necropsy observations of those animals found dead or sacrificed in a moribund condition were palpable masses and enlargement of the pituitary gland and the spleen, showing no influence with respect to treatment group.

BODY WEIGHT AND WEIGHT GAIN
Body weight development appeared normal when compared to concurrent and historical controls. No treatment-related effects were observed.

FOOD CONSUMPTION
Food consumption ranged from 15 to 20 g/day for males and from 12 to 16 g/day for female rats and showed no dependence upon treatment.


HAEMATOLOGY
The slightly elevated leukocyte count in blood in the silica group accompanied by increased neutrophils was indicative of an inflammatory reaction in the lungs.

CLINICAL CHEMISTRY
Measured virologic, bacteriologic, and parasitologic parameters were also within normal limits or negative during the study. Clinical laboratory test results at 6, 12, 18, and 25.5 months were essentially within normal range. Larger fluctuations in the values of the investigated clinical parameters at the end of the study were most likely due to age-related diseases.


ORGAN WEIGHTS
No changes in organ weights, except for the lungs and lung-associated lymph nodes were observed at the terminal sacrifice. Both the lung weight and lung weight to body weight ratio of the silica-exposed animals were elevated throughout the study. The lung weight of the silica-exposure group gradually increased throughout the study from 14% to 40% at 24 months compared to concurrent air-only controls.


HISTOPATHOLOGY: NON-NEOPLASTIC
Particle-laden macrophages were not seen in the SiO2-exposed rats, as the very small silica particles could be barely seen microscopically under polarizing light. The lungs of the silica-exposed rats showed a high incidence of granulomatous inflammatory foci, the extent of which increased with exposure duration.
Bronchiolar/alveolar hyperplasia of a slight to moderate degree was noted in the SiO2 group. Alveolar squamous cell metaplasias developed only in the lungs of both male and female silica-exposed rats. Alveolar lipoproteinosis of slight to moderate degree in the SiO2-exposed group was also reported at the study termination. Alveolar accumulation of foamy macrophages was observed. Both the incidence and severity of this finding increased with time for the SiO2-exposed group.
A pronounced and time-dependent increase in the severity of lung fibrosis was found in the SiO2-exposed group. A minimal to mild degree of fibrosis at 9 months, a mild degree at 15 months, and a mild to moderate degree at 21 months were observed in the SiO2-exposed group. A moderate degree of lung fibrosis was observed in 92% of the SiO2-exposed rats by the termination of the study. No significant difference from air-only controls in the extent of various upper respiratory system lesions was noted among the various treatment groups. Comparable frequencies of rhinitis, respiratory epithelial cell hyperplasia, goblet cell hyperplasia, and hyperplasia of submucosal glands were diagnosed among the treatment groups. The majority of these lesions could be related to the aspiration of food and thus are unlikely to be exposure related. Lymphoid hyperplasia of the lung-associated lymph nodes was observed in the SiO2-exposed group. Granulomatous lymphadenitis was also seen in all of the SiO2-exposed rats.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
An increased incidence of lung tumors was observed in the SiO2 exposed group. A total of 20 primary lung tumors were found in 19 animals. Two tumors, an adenoma and an adenocarcinoma, were observed in separate areas of the lung of one male silica-exposed rat. The distribution of tumor types consisted of 3 adenomas, 11 adenocarcinomas, 4 benign cystic keratinizing squamous cell tumors, 1 adenosquamous carcinoma, and 1 squamous cell carcinoma. There were also at least 13 proliferative lesions (nodular bronchiolar/alveolar hyperplasias), which may be thought of as preneoplastic changes in the same group. In addition to the above-cited tumors, three primary lung tumors were detected at the 21-month serial sacrifice. They consisted of two adenocarcinomas in the toner high-exposure group and one adenoma in the SiO2 exposed group, respectively.

Effect levels

Dose descriptor:
LOAEC
Effect level:
0.74 other: mg/m³ respirable aerosol concentration
Sex:
male/female
Basis for effect level:
other: organ weights; histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Particle retention

SiO2 accumulated progressively in the lungs of the rats. The mean quantities of toner retained in the lungs of the rats (combined male and female) at 24 months 0.91 mg/lung.

Bronchoalveolar Lavage (BAL)

The number of lavagable cells at 15 months of exposure was affected by treatment for the SiO2 exposure group where a 36% decrease was observed (see Table 4, refer to attached background material). A decrease in lavagable macrophages was evident in the SiO2-exposed group. In the lavage fluid a substantial amount of fragments of macrophages were observed. The levels of cytoplasmic and lysosomal enzymes and total protein in lavage fluid were significantly elevated for the SiO2 group, when first evaluated at 15 months of exposure. The mean level of LDH for the SiO2 group increased 16-fold compared to air-only controls. The corresponding values for /S-glucuronidase were elevated 53-fold. Finally the protein levels measured were 5.7 times higher than the corresponding control values at 15 months. The results at 21, 24, and 25.5 months showed similar elevation compared to controls.

Applicant's summary and conclusion

Conclusions:
A chronic inhalation study of crystalline silica (DQ-12) was conducted by exposure of groups of F-344 rats for 6 hr/day, 5 days/week for 24 months. Lung inflammation, a time-dependent increase in lung-to-body weight relative to control were observed. By the end of the study, 92.3% of the treated animals showed moderate lung fibrosis. Furthermore, primary lung tumors occurred in 19% of the silica-exposed rats, while tumor incidence occurred in 3% of the control animals. Survival was not affected by the particle treatment.