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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 01 NOV 1978 to 12 DEC 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (no GLP)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979

Materials and methods

Principles of method if other than guideline:
21-days repeated dose inhalation toxicity study
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: RAI f SPF (RA25)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: mean body weights males: 172-177g; mean body weights females: 165-171g
- Housing: groups of 5; Macrolon cages type 3
- Diet: pelleted standard diet, Nafag No. 890 (NAFAG, Gossau SG, Switzerland)
- Water: tap water, ad libitum
- Acclimatisation to exposure conditions: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 15

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: 70-80% < 7µm in diameter
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: in PVC-tubes which were positioned radially around the exposure chamber
- System of generating particulates/aerosols: by injecting three different amounts of the solid test material with the help of a Grafix Exaktomat Injector (Cerutti AG, Bern, Switzerland) into an airstream which was discharged into the exposure chamber
- Temperature, humidity, pressure in air chamber (group means): 23 °C, 50-56%, 2 atm
- Air flow rate: 20 l/min
- Method of particle size determination: gravimetrically with a 4 stage cascade impactor on selectron filters, pore size 0.2 µm and 25 mm diameter



TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically on selectron filters, pore size 0.2 µm and 50 mm in diameter
- Samples taken from breathing zone: yes
- Frequency: 5 times a day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
gravimetrically on selectron filters, pore size 0.2 µm and 50 mm in diameter
Duration of treatment / exposure:
21 days (15 exposure days), 6 hours/day
Frequency of treatment:
5 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 52, 151, 401 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations: daily, except weekends, during exposure; weekly during recovery


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: twice weekly during exposure; weekly during recovery


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: Yes


WATER CONSUMPTION: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all dose groups


HAEMATOLOGY: Yes
- Time schedule for collection of blood: once at the end of the exposure
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Parameters examined: haemoglobin, methaemoglobin, carboxyhaemoglobin, erythrocytes, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, reticulocytes, inclusion bodies, thrombocytes, prothrombin time, activated partial thromboplastin time, leucocytes (total count and differential count)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once at the end of the exposure
- Animals fasted: Yes
- How many animals: all
- Parameters examined: glucose, urea, total protein, protein electrophoresis, electrolytes, glutamate oxalacetate transaminase, glutamate pyruvate transaminase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl-transpeptidase


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
- organ weights: yes (at the end of the exposure and recovery period; brain, heart, liver, kidney,adrenals, thymus, gonades, lung)


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
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
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No death occurred during the exposure and recovery period. No toxic symptoms were observed in the animals of the control and test groups.

BODY WEIGHT AND WEIGHT GAIN
There was a slight but significant (p>/= 0.01) decrease in the male animals of the highest concentration group at day 21 and females at days 3, 7 and 10. During the recovery period the bodyweight gain was again comparabel to that of the control.

FOOD CONSUMPTION
No significant differences between treated animals and controls.

FOOD EFFICIENCY
No significant differences between treated animals and controls.

WATER CONSUMPTION
No effects.

OPHTHALMOSCOPIC EXAMINATION
No occular changes were observed.

HAEMATOLOGY
The observed haematological findings between treated rats and controls were generally unremarkable.
A slight change with statistical significance (p
CLINICAL CHEMISTRY
No significant differences between treated animals and controls.


ORGAN WEIGHTS
Absolute and relative lung weigths (lung to body weight and lung to brain weight) of males and females of the highest dose group were significantly elevated in comparison to the control group at the end of the exposure and at the end of the recovery period. Lung to body weights (but not the absolute lung weights) of males of the mid dose group were also elevated at the end of the exposure period (not measured at the end of the recovery period). The organ weights of the other organs were comparable in the control and treated groups.

GROSS PATHOLOGY
The lungs of all rats from the highest concentration group were slightly enlarged, with yellowish tinge. The lungs of all rats from the intermediate concentration group showed slightly yellowish tinge. The lungs of the lowest concentration group showed no compound related gross anatomical changes. The yellwoish discoloration of lungs was noted also in rats from the highest concentration group which were sacrificed after an additional recovery period of 21 days.

HISTOPATHOLOGY: NON-NEOPLASTIC
The eyes of all animals showed posttraumatic peribulbar lesions which were results of blood collection performed before sacrifice of the animals and were of no toxicologic relevance.
The lungs of all rats from the highest concentration group showed focal accumulation of slighly basophilic material and foamy cells in numerous alveoly. In Sudan stained frozen sections minute yellow-brown particles in the lumen of alveoli, in the cytoplasm of some foamy cells, in the lumen of occasional small bronchi and in the macrophages in the interstitium were seen. There was only minimal focal lymphohistiocytic infiltration in the interstitium.
The lungs of all rats from the intermediate concentration group showed , in the frozen section, moderate focal accumulation of brown-yellow minute particles in the macrophages in the interstitium and in very occasional alveoli. In the lungs of 4/10 male and 4/10 female rats from this group minimal focal accumulation of foamy cells in very occasional alveoli was observed.
The lungs of the rats from the lowest concentration goup showed no accumulation of foamy cells in the alveoli. In the frozen sections however minimal focal accumulation of brown-yellow particles in the macrophages in the interstitium and in very occasional alveoli was seen.
At the end of the recovery period the lungs of all animals from the highest concentration group showed focal accumulation of minute brown-yellow foreign particles in the interstitium. Few minute foreign particles in the alveoli and occasional ones in the intrapulmonal lymphatic tissue were observed as well.



Effect levels

Dose descriptor:
NOAEC
Effect level:
52 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: histopathologic investigation revealed minimal deposition of test material in the lung, but this was not accompanied by an inflammatory or any other adverse response

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Analysed test item concentrations were:

0 mg/m3

52 +/- 4 mg/m3

151 +/- 9 mg/m3

401 +/- 15 mg/m3

Applicant's summary and conclusion

Conclusions:
The test item did not elicit relevant toxicological effects after subacute inhalation exposure except for some local effects in the lungs of the treated animals probably due to the particulate matter of the test item and not due to inherent toxicity. Therefore, no NOEL can be derived from this study. Whereas inflammatory effects up to pneumoconiosis occurred in the mid and high concententration groups, the effects observed at the lowest test concentration were only due to the deposition of the test material, but not adverse. Therefore the lowest test concentration can be regarded as NOAEC.
Executive summary:

Subacute toxicity of the test item was investigated in an 21 days aerosol inhalation study in rats, which were exposed to 0, 52, 151, 401 mg/m3 test item (6 h/d, 5 d/w).

A slight but significant (p < 0.01) decrease in the bodyweight of the male rats an the highest concentration at termination of the exposure period was observed when compared with those of the controls and the other treated animals.

A slight change was found to occur in the differential leucocyte count of male and female rats at the highest exposure level, where a higher percentage of polymorphonuclear neutrophils and a lower percentage of lymphocytes was observed. This change persisted upon cessation of treatment.

The lungs of all rats from the highest concentration group were slightly enlarged and yellowish in colour. The yellowish dis­coloration of the lungs was also noted in rats from the group which was sacrificed after an additional recovery period of

21 days. The lung weights, the lung to bodyweight and lung to brain weight ratios of these animals were significantly increased. The weight increase persisted in rats of this group after anadditional recovery period of 21 days.

Upon histopathological examination the lungs of all rats from the highest concentration group showed focal accumulation of slightly basophilic material and of numerous foamy cells in the alveoli. In frozen sections large amount of minute yellow-brownforeign particles (1 - 3 um) in the lumen of the alveoli, in the cytoplasm of some foamy cells, in the lumen of occasional small bronchi and in the macrophages in the interstitium were observed. There was also slight focal lymphohistiocytic infiltration in the interstitium. Focal pneumoconiosis showing brown-yellow foreign particles (dissolved by the procedure used for tissue embedding) was also observed in the treated rats from the top concentration level after the withdrawal period of 3 weeks. Small accumulation of brown-yellow particles was occasionally found in the intra­pulmonal lymphoid tissue of these animals.

In the rats treated with the intermediate concentration level the lungs showed at autopsy slight yellowish discoloration. Histo­pathology revealed brown-yellow, fat soluble particles in the alveoli and in the interstitium of the lungs. In 8 out of 20 rats from this group focal accumulation of foamy cells in several alveoli was observed as well.

The yellowish discoloration of the lungs was not observed in rats from the lowest concentration group. Neither accumulation of foamy cells in the alveoli, nor interstitial inflammatory infiltration was seen upon histopathology in these animals. Minute brown­yellow fat soluble foreign particles were however found in frozen sections in the interstitium of the lungs and in occasional alveoli.

No other gross or microscopical changes which could be related to the inhalation of the test item were found in the treated animals.

It can be inferred from the observations made during the above study that the "no observable effect level" for rats is below 52 mg/m3of air. The effects observed at the lowest test concentration were only due to the deposition of the test material but did not cause adverse effects like inflammation etc. Therefore, the lowest test concentration can be regarded as "no observable adverse effect level".