Ashes (residues), coal

Acute Toxicity: other routes

Administrative data

Endpoint:

acute toxicity: other routes

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 Aug 1992 - 25 Sep 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented study report which meets basic scientific principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

acute toxicity: other routes

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 Aug 1992 - 25 Sep 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented study report which meets basic scientific principles.

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

The respiratory tract response to fly ash "Maasvlakt" was studied in rats after a single intratracheal instillation of the test materials at 3 different concentrations. Analysis of bronchoalveolar lavage was carried out at intervals up to 28 days post-application. The remaining rats at the end of the study were examined histopathologically.

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatsoezicht op de volksgezondheid, Veterinaire hoofdinspectie (Central Veterinary Publich Health Inspectorate), Riswijk, The Netherlands

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: ca. 9 weeks
- Weight at study initiation: ca. 250 g
- Housing:The rats were housed in groups of four from day -4 until autopsy, except for the rats of the second saline control group which were housed three per cage. The rats of each group to be sacrificed at the various time points were taken from different cages. Consequently, the number of rats per cage decreased in the course of the study. The rats were kept in suspended, stainless cages, fitted with wire mesh floor ans front.
- Diet (e.g. ad libitum): Institute's cereal-based, powdered stock diet for rats, mice and hamsters ad libitum
- Water (e.g. ad libitum): Local commuity tap-water ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21.5
- Humidity (%): 57-75
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: August 18, 1992 To: September 25, 1992

Route of administration:

other: intratracheal instillation

Vehicle:

physiological saline

Details on exposure:

The test material was given at three dose levels; silicon dioxide (positive control) and titanium dioxide (negative control) were given at two dose levels. The highest dose of silicon dioxide was intended to result in toxic effects but should not produce an incidence of fatalities which would prevent a meaningful evaluation. The lowest dose was intended to provide a minimum-toxic-effect level of this substance. Titanium dioxide served as a negative control and was given at the same two levels to provide a similar particle (over)load. The test substance was given at the same those levels plus an additional lower level in order to compare the effects with both the positive and negative control.
Since the total number of rats to be dosed and sacrificed on a day was too large, dosing and experimental handlings were spread over two days. Two control groups were used, one on either day, in order to rule out day-by-day variations. One saline control and the two negative and two positive control groups were treated on one day (day 0 (dosing), and 7, 14, 28 (lavage studies)), the other saline control and the six test groups were treated on the second day (day 1 (dosing), and 8, 15, 29 (lavage studies)).
The test substance was heated to 180°C for 1-2 h for sterilisation prior to use, suspended in sterile saline, homogenised using an ultrasonic bath, and were administered as suspensions by a single intratracheal injection under ether anaesthesia. Rats of different groups were dosed with different concentrations of the test substance in saline, to ensure a constant dose volume per kg bw at all dose levels. The dose volume was 1 mL/kg bw. Controls were treated with saline only. The route of administration was chosen to mimic the usual route of human exposure.

Doses:

Control: 0 mg/kg bw
Fly ash "Maasvlakte": 2, 10, 50 mg/kg bw
Silicon dioxide: 10, 50 mg/kg bw
Titanium dioxide: 10, 50 mg/kg bw

No. of animals per sex per dose:

16 males per dose

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 7, 14, 28 days (4 animals per dose were used for histopathology and sacrificed on day 31 post-administration)
- Frequency of observations and weighing: The body weight of each animals was recorded twice during the acclimatisation period (days -4 and -1), when starting the administration of the test substance and once every week thereafter. In addition, the rats intended for histopathology were weighed on the day of sacrifice.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight (gain), lung weights, lung histopathology, lavage fluid analysis (total protein, lactate dehydrogenase [LDH], beta-glucuronidase, N-acetyl-beta-D-glucosaminidase [NAG])

Statistics:

Body weight data were analysed by One-way Analysis of Covariance (ANCOVA) using pre-exposure (day 0) weights as covariate, followed by Student t-tests (two-tailed). Growth rate and data on organ weights were analysed by One-way Analysis of Variance (ANOVA) followed by Dunnett’s Multiple Comparison Tests (two-tailed). For both body weight and growth rate analysis, the joined saline control group acteed as the control group. For analysis of organ weights the first saline control group acted as the control group.
Incidences of histopathological changes were analysed by Fischer’s exact probability test.
Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant.
In addition, Jonckheere’s trend test and Mann-Whitney U-test were applied to establish a statistical no-effect-level for increase in both cellular (absolute numbers) and biochemical markers in lung lavage fluid for each compound. The test procedure included two steps: at step I all groups were tested by Jonkheere’s trend test; if the result was significant (p < 0.05, one-tailed) the test was repeated without the highest dose group until a non-significant result occurred. At step II pairwise comparisons with the control and the remaining dose groups were made. Fro the results of step I and II, a statistical no-effect-level was determined. The joint saline control group acted as the control group.

Sex:

male

Dose descriptor:

other: NOEL

Effect level:

2 mg/kg bw

Remarks on result:

other: based on bronchoalveolar fluid analysis and histopathological changes

Mortality:

No mortalities occurred.

Clinical signs:

No effects related to treatment with the test material were observed. One silicon dioxide-treated rat (50 mg/kg) showed alopecia areas on its fur and an abnormal appereance of the head from day 21-28.

Body weight:

Compared to control rats, body weights were significantly decreased in rats treated with 50 mg/kg bw silicon dioxide during the whole test period, and in rats treated with 50 mg/kg bw titanium dioxide or fly ash "Maasvlakte" until day 28. The decreases in body weight were caused by a strong, significant decrease in body weight gain during the first test week, which was not caught up with the controls during the remainder of the test period. A decreased body weight gain was also seen in rats treated with 50 mg/kg bw silicon dioxide between days 21 and 28.
In addition, body weight was decreased in rats treated with 10 mg/kg bw fly ash "Maasvlakte" on day 14.

Gross pathology:

Treatment-related gross changes were observed in the lungs and mediastinal lymph nodes of almost all animals treated with either 50 mg/kg bw silicon dioxide, titanium dioxide or fly ash "Maasvlakte". These changes consisted of discolouration and/or enlargement of the mediastinal lymph node and spotted, discoloured, swollen and/or nodular lungs. Furthermore, also in rats treated with 10 mg/kg bw silicon dioxide, 3 out of 4 animals showed macroscopic changes in lungs and mediastinal lymph nodes.
All other gross changes such as enlarged cervical lymph nodes, small testes and spotted thymus occurred in a single animal only. They were, therefore, considered to be toxicologically unimportant.

Other findings:

- Lung weights: Absolute and relative lung weights were significantly increased in rats treated with silicon dioxide. A dose-response relationship was observed. In the other groups, no significant changes in lung weight compared with controls was observed.
- Histopathology: Microscopic examination of the lungs exhibited multifocal accumulation of particulate material in the alveoli of most animals treated with the various test compounds.
All animals given silicon dioxide except one, showed treatment-related pulmonary histopathological changes which were characterized by perivascular lymphoid aggregates, accumulation of alveolar macrophages, increased septal cellularity, polymorphonuclear leukocyte infiltration and small granulomas in peribronchial lymphoid tissue. Moreover, all 50 mg/kg bw silicon dioxide animals developed multiple granulomas in alveolar tissue.
Treatment-related histopathological pulmonary changes observed in rats treated with 50 mg/kg bw titanium dioxide comprised increased septal cellularity and perivascular lymphoid aggregates. The test compound was visible in peribronchial lymphoid tissue in the form of fine brown particles. In the 10 mg/kg bw titanium dioxide group, only one animal showed perivascular lymphoid aggregates.
Two out of 4 animals treated with 2 mg/kg bw fly ash "Maasvlakte" developed very slight perivascular lymphoid aggregates and one of them additionally showed a minimal accumulation of alveolar macrophages. Most animals given 10 or 50 mg/kg bw fly ash "Maasvlakte" exhibited perivascular lymphoid aggregates, accumulation of alveolar macrophages and increased septal cellularity, which, in the 50 mg/kg bw group, was accompanied by the devolpement of multiple granulomas in 2 out of 4 rats exposed to fly ash "Maasvlakte".
In the mediastinal lymph nodes, accumulation of the test compound was visible in all animals exposed to 10 or 50 mg/kg bw titanium dioxide and in 3 out of 4 animals exposed to 50 mg/kg bw fly ash "Maasvlakte". The mediastinal lymph nodes of the fly ash "Maasvlakte"-treated rats exhibited an "activated" appearance. Multiple granulomas developed in the mediastinal lymph nodes of most rats exposed to silicon dioxide.
The histopathological changes observed in the lungs and mediastinal lymph nodes of the groups treated with either 10 or 50 mg/kg bw fly ash "Maasvlakte" have been considered to be related to the treatment, since these changes are uncommon findings in animals of this strain and age.
- Potential target organs: lung and mediastinal lymph nodes
- Lavage fluid analysis: Compared to controls, total cell count was significantly increased in lung lavage fluid of all rats treated with silicon dioxide and in rats treated with 50 mg/kg bw of each of the other compounds at all sampling days. Highest numbers were always observed in silicon dioxide-treated rats reaching significant differences with each of the other groups at all time points except on day 7 on which rats treated with either 50 mg/kg bw silicon dioxide or titanium dioxide showed no significant difference. Increased total cell count was also observed in rats treated with 10 mg/kg bw fly ash "Maasvlakte" seven days after instillation. The number of total cells seemed to decrease with time except for the 10 mg/kg bw silicon dioxide group.
The mean number of viable cells in the lavage was between 87 and 97%.
The relative number of monocytes/macrophages decreased and the relative numbers of granulocytes and lymphocyte increased in all 10 and 50 mg/kg bw treatment groups at all sampling days; groups treated with 2 mg/kg bw fly ash"Maasvlakte" showed no differences to controls.
Compared to control rats, absolute numbers of monocytes/macrophages were increased in rats dosed with silicon dioxide (all sampling days) and in rats treated with 50 mg/kg bw titanium dioxide (day 7). Statistically significant differences between corresponding dose groups were only observed at day 14 (10 mg/kg bw silicon dioxide and fly ash "Maasvlakte") and at day 28 (10 mg/kg silicon dioxide and either other compound; 50 mg/kg bw silicon dioxide and titanium dioxide).
Absolute numbers of granulocytes and lymphocytes were generally higher in silicon dioxide-treated rats compared to controls or with rats treated with either other compound at a corresponding dose level at all sampling days. The level of statistical significance between animals given 50 mg/kg bw silicon dioxide and those treated with 50 mg/kg bw of each of the other compunds was not reached in lymphocytes numbers on days 7 and 14, which was explained by the decreased number of rats (3 instead of 4) in the 50 mg/kg bw silicon dioxide group on those days.
Furthermore, there are common points of interest:
(1) On day 28, lymphocyte numbers of 50 mg/kg bw fly ash "Maasvlakte"-treated animals were significantly higher than in rats treated with 50 mg/kg bw titanium dioxide. The lymphocyte numbers of the fly ash "Maasvlakte" and silicon dioxide-treated groups seemed to increase with time.
(2) Rats treated with 2 mg/kg bw fly ash "Maasvlakte" did not show significantly different lymphocyte numbers.
(3) The numbers of granulocytes tended to decrease slightly with time except for the 10 mg/kg bw silicon dioxide group which showed a steep, and the 50 mg/kg bw fly ash "Maasvlakte" group that showed a slight time-related increase.
(4) Using trend analysis, a very slight, though significant, increase in granulocyte numbers was observed in rats given 2 mg/kg bw fly ash "Maasvlakte" on days 14 and 28.

Protein content in lung lavage fluid was higher in rats treated with silicon dioxide or with 50 mg/kg bw titanium dioxide than in control rats at any time point. In addition, silicon dioxide-treated groups showed increased protein content compared to the other treatment groups at corresponding dose levels. Rats treated with fly ash "Maasvlakte" showed increased levels at doses of 10 or 50 mg/kg bw varying per sample day. Protein levels generally seemed to decrease with time except for silicon dioxide-treated groups.
Compared to control rats, LDH, NAG and beta-glucuronidase activities were increased in rats treated with silicon dioxide. The increases were dose-related and a time-relationship was observed in the 10 mg/kg bw group.
Compared to the other treatment groups, these activities were also higher in silicon dioxide-dosed rats, except for the 50 mg/kg bw groups on day 14 (all activities) and on day 28 (LDH activity only). At some other occasions, the level of statistical significance was not reached either. This was explained by decreased group size on day 7 (3 instead of 4 rats in the 50 mg/kg bw silicon dioxide group) or low values in general on day 14 (50 mg/kg bw silicon dioxide group).
In rats treated with titanium dioxide, LDH was increased in the 50 mg/kg bw group at all sampling days; NAG and beta-glucuronidase activity were not adversely affected except for an increased beta-glucuronidase activity in 50 mg/kg bw treated rats on day 28.
Treatment with fly ash "Maasvlakte" showed comparable results with regard to NAG activity (no changes at any level tested) and beta-glucuronidase activity (increased at 50 mg/kg bw in general) or slightly varying results with respect to LDH activity: LDH activity was increased in 50 mg/kg bw fly ash "Maasvlakte"-treated rats at all days and in 10 mg/kg bw rats on days 14. Time-response relationships were not observed.
LDH, NAG and beta-glucuronidase activities did, however, not differ between titanium dioxide and fly ash "Maasvlakte" dosed groups at corresponding dose levels. In addition, rats treated with 2 mg/kg bw fly ash "Maasvlakte" did not show any changes.

Conclusions:

From the results of the present study it was concluded that:
(1) Silicon dioxide treatment at doses of 10 and 50 mg/kg bw induced dose-related lung injury, which might result in progressive lung toxicity at these dose levels and higher,
(2) The relatively innocuous dust titanium dioxide induced pulmonary responses at a dose of 50 mg/kg bw which might be explained by dust overload mechanisms,
(3) Fly ash “Maasvlakte” induced effects more or less identical to those of titanium dioxide except for increased lymphocyte numbers at levels of 50 mg/kg bw especially towards the end of the study period (day 28), indicating that on-going (immunological) reactions may exist,
(4) With respect to the adverse effects observed at 10 and 50 mg/kg bw, the no-observed-effect level of fly ash “Maasvlakte” is 2 mg/kg bw.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Principles of method if other than guideline:

The respiratory tract response to lytag powder was studied in rats after a single intratracheal instillation of the test materials at 3 different concentrations. Analysis of bronchoalveolar lavage was carried out at intervals up to 28 days post-application. The remaining rats at the end of the study were examined histopathologically.

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatsoezicht op de volksgezondheid, Veterinaire hoofdinspectie (Central Veterinary Publich Health Inspectorate), Riswijk, The Netherlands

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues), coal

EC Number:

931-322-8

Cas Number:

68131-74-8

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Ashes (residues), coal

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: ca. 9 weeks
- Weight at study initiation: ca. 250 g
- Housing:The rats were housed in groups of four from day -4 until autopsy, except for the rats of the second saline control group which were housed three per cage. The rats of each group to be sacrificed at the various time points were taken from different cages. Consequently, the number of rats per cage decreased in the course of the study. The rats were kept in suspended, stainless cages, fitted with wire mesh floor ans front.
- Diet (e.g. ad libitum): Institute's cereal-based, powdered stock diet for rats, mice and hamsters ad libitum
- Water (e.g. ad libitum): Local commuity tap-water ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21.5
- Humidity (%): 57-75
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: August 18, 1992 To: September 25, 1992

Administration / exposure

Route of administration:

other: intratracheal instillation

Vehicle:

physiological saline

Details on exposure:

The test material was given at three dose levels; silicon dioxide (positive control) and titanium dioxide (negative control) were given at two dose levels. The highest dose of silicon dioxide was intended to result in toxic effects but should not have produced an incidence of fatalities which would prevent a meaningful evaluation. The lowest dose was intended to provide a minimum-toxic-effect level of this substance. Titanium dioxide served as a negative control and was given at the same two levels to provide a similar particle (over)load. The test substance was given at the same those levels plus an additional lower level in order to compare the effects with both the positive and negative control.
Since the total number of rats to be dosed and sacrificed on a day was too large, dosing and experimental handlings were spread over two days. Two control groups were used, one on either day, in order to rule out day-by-day variations. One saline control and the two negative and two positive control groups were treated on one day (day 0 (dosing), and 7, 14, 28 (lavage studies)), the other saline control and the six test groups were treated on the second day (day 1 (dosing), and 8, 15, 29 (lavage studies)).
The test substance was heated to 180°C for 1-2 h for sterilisation prior to use, suspended in sterile saline, homogenised using an ultrasonic bath, and were administered as suspensions by a single intratracheal injection under ether anaesthesia. Rats of different groups were dosed with different concentrations of the test substance in saline, to ensure a constant dose volume per kg bw at all dose levels. The dose volume was 1 mL/kg bw. Controls were treated with saline only. The route of administration was chosen to mimic the usual route of human exposure.

Doses:

Control: 0 mg/kg bw
Lytag powder: 2, 10, 50 mg/kg bw
Silicon dioxide: 10, 50 mg/kg bw
Titanium dioxide: 10, 50 mg/kg bw

No. of animals per sex per dose:

16 males per dose

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 7, 14, 28 days (4 animals per dose were used for histopathology and sacrificed on day 31 post-administration)
- Frequency of observations and weighing: The body weight of each animals was recorded twice during the acclimatisation period (days -4 and -1), when starting the administration of the test substance and once every week thereafter. In addition, the rats intended for histopathology were weighed on the day of sacrifice.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight (gain), lung weights, lung histopathology, lavage fluid analysis (total protein, lactate dehydrogenase [LDH], beta-glucuronidase, N-acetyl-beta-D-glucosaminidase [NAG])

Statistics:

Body weight data were analysed by One-way Analysis of Covariance (ANCOVA) using pre-exposure (day 0) weights as covariate, followed by Student t-tests (two-tailed). Growth rate and data on organ weights were analysed by One-way Analysis of Variance (ANOVA) followed by Dunnett’s Multiple Comparison Tests (two-tailed). For both body weight and growth rate analysis, the joined saline control group acteed as the control group. For analysis of organ weights the first saline control group acted as the control group.
Incidences of histopathological changes were analysed by Fischer’s exact probability test.
Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant.
In addition, Jonckheere’s trend test and Mann-Whitney U-test were applied to establish a statistical no-effect-level for increase in both cellular (absolute numbers) and biochemical markers in lung lavage fluid for each compound. The test procedure included two steps: at step I all groups were tested by Jonkheere’s trend test; if the result was significant (p < 0.05, one-tailed) the test was repeated without the highest dose group until a non-significant result occurred. At step II pairwise comparisons with the control and the remaining dose groups were made. From the results of step I and II, a statistical no-effect-level was determined. The joint saline control group acted as the control group.

Results and discussion

Effect levels

Sex:

male

Dose descriptor:

other: NOEL

Effect level:

2 mg/kg bw

Remarks on result:

other: based on bronchoalveolar fluid analysis and histopatholigical changes

Mortality:

No mortalities occurred.

Clinical signs:

No effects related to treatment with the test material were observed. One silicon dioxide-treated rat (50 mg/kg) showed alopecia areas on its fur and an abnormal appereance of the head from day 21-28.

Body weight:

Compared to control rats, body weights were significantly decreased in rats treated with 50 mg/kg bw silicon dioxide during the whole test period, and in rats treated with 50 mg/kg bw titanium dioxide and in rats treated with 50 mg/kg bw lytag powder in the first part of the test period (until day 14). The decreases in body weight were caused by a strong, significant decrease in body weight gain during the first test week, which was not caught up with the controls during the remainder of the test period except for lytag powder treated rats. A decreased body weight gain was also seen in rats treated with 50 mg/kg bw silicon dioxide between days 21 and 28.
In addition, body weight was decreased in rats treated with 10 mg/kg bw lytag powder on days 7 and 14. The decrease was explained by the decrease in body weight gain as observed on day 7.

Gross pathology:

Treatment-related gross changes were observed in the lungs and mediastinal lymph nodes of almost all animals treated with either 50 mg/kg bw silicon dioxide, titanium dioxide or lytag powder. These changes consisted of discolouration and/or enlargement of the mediastinal lymph node and spotted, discoloured, swollen and/or nodular lungs. Furthermore, also in rats treated with 10 mg/kg bw silicon dioxide, 3 out of 4 animals showed macroscopic changes in lungs and mediastinal lymph nodes.
All other gross changes such as enlarged cervical lymph nodes, small testes and spotted thymus occurred in a single animal only. They were, therefore, considered to be toxicologically unimportant.

Other findings:

- Lung weights: Absolute and relative lung weights were significantly increased in rats treated with silicon dioxide. A dose-response relationship was observed. In the other groups, no significant changes in lung weight compared to controls was observed.
- Histopathology: Microscopic examination of the lungs exhibited multifocal accumulation of particulate material in the alveoli of most animals treated with the various test compounds.
All animals given silicon dioxide except one, showed treatment-related pulmonary histopathological changes which were characterized by perivascular lymphoid aggregates, accumulation of alveolar macrophages, increased septal cellularity, polymorphonuclear leukocyte infiltration and small granulomas in peribronchial lymphoid tissue. Moreover, all 50 mg/kg bw silicon dioxide animals developed multiple granulomas in alveolar tissue.
Treatment-related histopathological pulmonary changes observed in rats treated with 50 mg/kg bw titanium dioxide comprised increased septal cellularity and perivascular lymphoid aggregates. The test compound was visible in peribronchial lymphoid tissue in the form of fine brown particles. In the 10 mg/kg bw titanium dioxide group, only one animal showed perivascular lymphoid aggregates.
Animals treated with 2 mg/kg bw lytag powder developed no histopathological changes. Most animals given 10 or 50 mg/kg bw lytag powder exhibited perivascular lymphoid aggregates, accumulation of alveolar macrophages and increased septal cellularity, which, in the 50 mg/kg bw group, was accompanied by the devolpement of multiple granulomas in 3 out of 4 rats exposed to lytag powder.
In the mediastinal lymph nodes, accumulation of the test compound was visible in all animals exposed to 10 or 50 mg/kg bw titanium dioxide and 50 mg/kg bw lytag powder. Multiple granulomas developed in the mediastinal lymph nodes of most rats exposed to silicon dioxide.
The histopathoilogical changes observed in the lungs and mediastinal lymph nodes of the groups treated with either 10 or 50 mg/kg bw lytag powder have been considered to be related to the treatment, since these changes are uncommon findings in animals of this strain and age.
- Potential target organs: lung and mediastinal lymph nodes
- Lavage fluid analysis: Compared to controls, total cell count was significantly increased in lung lavage fluid of all rats treated with silicon dioxide and in rats treated with 50 mg/kg bw of each of the other compounds at all sampling days. Highest numbers were always observed in silicon dioxide-treated rats reaching significant differences with each of the other groups at all time points except on day 7 on which rats treated with either 50 mg/kg bw silicon dioxide or titanium dioxide showed no significant difference. The number of total cells seemed to decrease with time except for the 10 mg/kg bw silicon dioxide group.
The mean number of viable cells in the lavage was between 87 and 97%.
The relative number of monocytes/macrophages decreased and the relative numbers of granulocytes and lymphocyte increased in all 10 and 50 mg/kg bw treatment groups at all sampling days; groups treated with 2 mg/kg bw lytag powder showed no differences to controls.
Compared to control rats, absolute numbers of monocytes/macrophages were increased in rats dosed with silicon dioxide (all sampling days), in rats treated with 50 mg/kg bw titanium dioxide (day 7) and in rats given 50 mg/kg bw lytag powder (days 7 and 14). Statistically significant differences between corresponding dose groups were observed at day 28 (10 mg/kg silicon dioxide and either other compound; 50 mg/kg bw silicon dioxide and titanium dioxide).
Absolute numbers of granulocytes and lymphocytes were generally higher in silicon dioxide-treated rats compared to controls or with rats treated with either other compound at a corresponding dose level at all sampling days. The level of statistical significance between animals given 50 mg/kg bw silicon dioxide and those treated with 50 mg/kg bw of each of the other compunds was not reached in lymphocytes numbers on days 7 and 14, which was explained by the decreased number of rats (3 instead of 4) in the 50 mg/kg bw silicon dioxide group on those days.
Furthermore, there are common points of interest:
(1) On day 28, an increased number of lymphocytes was observed in rats treated with 10 or 50 mg/kg bw lytag powder compared to controls; moreover, the numbers were not significantly diferent from rats treated with corresponding dose levels of silicon dioxide. In addition, lymphocyte numbers of the 50 mg/kg bw lytag powder-treated group were significantly higher than in rats treated with 50 mg/kg bw titanium dioxide on that day. The lymphocyte numbers of the silicon dioxide-treated groups seemed to increase with time.
(2) Rats treated with 2 mg/kg bw lytag powder did not show significantly different lymphocyte numbers.
(3) The numbers of granulocytes tended to decrease slightly with time except for the 10 mg/kg bw silicon dioxide group which showed a steep time-related increase.

Protein content in lung lavage fluid was higher in rats treated with silicon dioxide or with 50 mg/kg bw titanium dioxid ethan in control rats at any time point. In addition, silicon dioxide-treated groups showed increased protein content compared to the other treatment groups at corresponding dose levels. Rats treated with lytag powder showed increased levels at doses of 10 or 50 mg/kg bw varying per sample day. Protein levels generally seemed to decrease with time except for silicon dioxide-treated groups.
Compared to control rats, LDH, NAG and beta-glucuronidase activities were increased in rats treated with silicon dioxide. The increases were dose-related and a time-relationship was observed in the 10 mg/kg bw group.
Compared to the other treatment groups, these activities were also higher in silicon dioxide-dosed rats, except for the 50 mg/kg bw groups on day 14 (all activities) and on day 28 (LDH activity only). At some other occasions, the level of statistical significance was not reached either. This was explained by decreased group size on day 7 (3 instead of 4 rats in the 50 mg/kg bw silicon dioxide group) or low values in general on day 14 (50 mg/kg bw silicon dioxide group).
In rats treated with titanium dioxide, LDH was increased in the 50 mg/kg bw group at all sampling days; NAG and beta-glucuronidase activity were not adversely affected except for an increased beta-glucuronidase activity in 50 mg/kg bw treated rats on day 28.
Treatment with lytag powder showed comparable results with regard to NAG activity (no changes at any level tested) and beta-glucuronidase activity (increased at 50 mg/kg bw in general) or slightly varying results with respect to LDH activity: a significant increase in LDH activity was observed in 10 and 50 mg/kg bw lytag powder groups on day 14 only.
LDH, NAG and beta-glucuronidase activities did, however, not differ between titanium dioxide and lytag powder dosed groups at corresponding dose levels. In addition, rats treated with 2 mg/kg bw lytag powder did not show any changes.

Applicant's summary and conclusion

Conclusions:

From the results of the present study it was concluded that:
(1) Silicon dioxide treatment at doses of 10 and 50 mg/kg bw induced dose-related lung injury, which might result in progressive lung toxicity at these dose levels and higher,
(2) The relatively innocuous dust titanium dioxide induced pulmonary responses at a dose of 50 mg/kg bw which might be explained by dust overload mechanisms,
(3) Lytag powder induced effects more or less identical to those of titanium dioxide except for increased lymphocyte numbers at levels of 50 mg/kg bw especially towards the end of the study period (day 28), indicating that on-going (immunological) reactions may exist,
(4) With respect to the adverse effects observed at 10 and 50 mg/kg bw, the no-observed-effect level of lytag powder is 2 mg/kg bw.