Registration Dossier

Administrative data

short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 March 2002 to 30 October 2002
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
Limit test:

Test material

Test material form:
solid: particulate/powder
Details on test material:
- Physical state: solid
- Storage condition of test material: room temperature

Test animals

other: Crl:CD(SD)IGS BR
Details on test animals and environmental conditions:
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: 175-198 g (males); 165-184 g (females)
- Housing: individually in suspended, stainless steel, wire-mesh type cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

- Temperature: 66-73 °F
- Humidity: 30-58 %
- Photoperiod: approximately 12 hours fluorescent light per day

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Remarks on MMAD:
2.3 mg/M3 - 1.64 ± 0.169 µ/M; 5.1 mg/M3 - 2.11 ± 0.165 µ/M; 12.0 mg/M3 - 1.62 ± 0.113 µ/M

Mean GSD
2.3 mg/M3 - 1.653 ± 0.223; 5.1 mg/M3 - 1.753 ± 0.332 µ/M; 12.0 mg/M3 - 1.620 ± 0.136 µ/M
Details on inhalation exposure:
- Exposure apparatus: 63 L stainless steel and acrylic nose-only exposure chamber with stainless steel baffle (see Figure below).
- Method of holding animals in test chamber: nose-only restraint tubes
- System of generating particulates/aerosols: test substance was packed into a cylindrical holder with high pressure to provide a compact, uniform surface. A scraping blade in a Wright Dust Feeder (WDF) rotated over the surface removing small amounts of the test substance. The dislodged test substance entered the air stream passing through the WDF and was transported out of the WDF into the jet mill. The jet mill pulverized the test substance by directing air streams containing the test substance particles into each other from opposing directions. Smaller pulverized particles exited the jet mill whereas larger particles went through the pulverization process again. The test substance then passed through a cyclone which further separated out the larger particles from the aerosol prior to entering the exposure chamber.
- Temperature, humidity, pressure in air chamber: 20-24 °C; relative humidity 40-60 %
- Air flow rate: 83, 82, 70 and 68 L/min for controls, low dose, mid dose and high dose groups respectively.
- Air change rate: at least 64 per hour
- Method of particle size determination: particles were accelerated through a nozzle in a TSI Aerodynamic Particle Sizer and measuring the time taken for each particle to pass between two closely spaced laser beams
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Chamber atmosphere samples for the gravimetric determination of the test material exposure levels were collected. The samples were withdrawn from the breathing zone of the animals in the exposure chambers through glass-fibre filters.

The gravimetric test material exposure levels were measured at least twice during the exposure. The aerosol concentration was calculated as the filter weight gain in mg divided by the volume of air sampled. The volume of air sampled was calculated as the sample flow rate multiplied by the sample duration.

The samples collected were transferred to polypropylene jars, diluted with HCl solution and analysed with HPLC.
Duration of treatment / exposure:
Six hours
Frequency of treatment:
Once per day, 5 days/week for 20 exposure days
Doses / concentrationsopen allclose all
Dose / conc.:
2.3 mg/m³ air (analytical)
Dose / conc.:
5.1 mg/m³ air (analytical)
Dose / conc.:
12 mg/m³ air (analytical)
No. of animals per sex per dose:
Control animals:


Observations and examinations performed and frequency:
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes - evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling, bizarre behaviour and palpatation of tissue masses.
- Time schedule: weekly

- Time schedule for examinations: weekly

- Food consumption: Yes - weekly

- Time schedule for examinations: prior to the start of the test and again prior to termination

- Time schedule for collection of blood: at termination of the study
- Anaesthetic used for blood collection: Yes, carbon dioxide
- Animals fasted: Yes
- How many animals: all
- Parameters checked: haemoglobin, erythrocyte count, absolute and percent reticulocytes, platelets, prothrombin time, activated partial thromboplastin time, total and differential leucocyte counts, hematocrit, MCV, MCH and MCHC.

- Time schedule for collection of blood: at termination of the study
- Animals fasted: Yes
- How many animals: all
- Parameters checked: sodium, potassium, chloride, calcium, phosphorus, glucose, urea nitrogen, cholesterol, total bilirubin, total protein, albumin, globulin, albumin/globulin ratio, creatinine, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase and sorbitol dehydrogenase.

- Time schedule for examinations: prior to termination of the study
- Dose groups that were examined: all
- Battery of functions tested: motor activity / functional observational battery
Sacrifice and pathology:
- The following organs were weighed: adrenals, brain, kidneys, liver, heart, lungs, pituitary, ovaries, prostate, spleen, testes, thymus, thyroids and uterus.

- The following organs/tissues were sampled: adrenals, aorta, sternum with bone marrow, femur with bone marrow, bone marrow smears, brain, caecum, colon, duodenum, epididymis, eyes, harderian gland, heart, ileum, jejunum, kidneys, lacrimal gland, larynx, liver, lungs, mandibular and mesenteric lymph nodes, mammary gland (females only), nasal tissues, oesophagus, optic nerves, ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerves, seminal vesicle, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus, vagina and all gross lesions.
Levene's test was used to assess homogeneity of group variances. If Levene's test was not significant (p≥0.01)< Dunnett's test was used to compare each treatment group with the control group. If Levene's test was significant (p<0.01), comparisons with the control group were made using Welch's t-test with Bonferroni correction.

Chi-square test for homogeneity was used to analyse Functional Observational Battery results except for continuous data.

Log transformation was performed on leucocyte counts prior to analysis.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test material related clinical observations seen in the study.
no mortality observed
Description (incidence):
No unscheduled deaths occurred during the course of the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no clear test material effect on the body weight. The mean weight for the females in the highest dose group was statistically lower than the control mean weights. However, this decrease appeared to be due to one animal, therefore this is not clearly test material related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no test material related effect on food consumption. The statistically significant differences that occurred in some weeks were sporadic and showed no relationship to dose.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test material related Ophthalmoscopic effects were seen.
Haematological findings:
no effects observed
Description (incidence and severity):
When compared to controls, no toxic effects on haematology were observed after 28 days for male or female rats exposed up to the highest dose level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related toxic effects on clinical chemistry parameters were observed for males or females exposed up to the highest dose level. Any observed alterations were not dose dependent and were considered related to inter-day collection variety.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Motor activity evaluations revealed no test material related effects. There was no test material related effect seen on any of the functional observational categories of activity/arousal, neuromuscular, sensorimotor, autonomic, or physiological measurements. The only statistically significant differences seen in the highest dose group was in hind limb grip strength and body temperature in the males. No difference was seen in fore limb strength and neither of these differences was seen in the females. These differences were therefore considered to be spurious and not related to the test material.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test material related organ weight changes occurred in either sex.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test material related macroscopic observations were noted in either sex.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test material related microscopic effects noted in either sex.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
Effect level:
12 mg/m³ air
Based on:
test mat.
Basis for effect level:
other: No test material related effects were seen.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean body weights (g)

     Dose group (mg/mm3)
 Time (weeks)  Sex  0.0  2.3  5.1  12.0
 -1  M  183.0  192.2*  194.3*  180.1
 1  M  233.8  237.1  240.0  229.7
 2  M  282.9  278.8  280.5  277.6
 3  M  318.9  309.6  309.3  312.6
 4  M  337.2  324.5  326.0  333.0
 -1  F  169.4  173.7  172.3  169.3
 1  F  192.4  188.3  192.8  185.3
 2  F  209.5  201.9  207.0  198.5
 3  F  226.1  214.2  222.1  209.9*
 4  F  232.1  222.7  228.3  216.8

*significantly different from control (P<0.05)

Table 2: Selected haemotology values

     Dose group (mg/m3)
 Parameter  Sex  0.0  2.3  5.1  12.0
 Platelets (K/mm3)  M  1205.0  1013.2  921.1**  1045.7
   F  1033.3  1089.5  879.9  1139.1
 APTT (sec)  M  30.18  19.38**  19.58**  31.00
   F  23.44  16.93**  17.47**  21.50
 Prothrombin time (sec)  M  17.67  14.80**  14.65**  17.27
   F  13.98  14.64  14.14  13.66

**significantly different from control (P<0.01)

Table 3: Selected clinical chemistry values

     Dose group (mg/m3)
 Parameter  Sex  0.0  2.3  5.1  12.0
 Sodium (mEq/L)  M  148.2  151.2**  150.8**  149.0
   F  146.3  148.6  149.2*  147.5
 Potassium (mEq/L)  M  7.18  6.11  5.99*  6.98
   F  7.44  6.52  5.97**  7.50
 Sorbital dehydrogenase (U/L)  M  16.11  23.58**  23.99**  15.77
   F  16.51  23.25*  21.87  16.88
 Cholestoerol (mg/dL)  M  47.1  57.9  59.2*  48.3
   F  53.8  66.2  7.1**  60.6
 Glucose (mg/dL)  M  10.64  90.8*  100.3  110.6

*significantly different from control (P<0.05)

**significantly different from control (P<0.01)

Table 4: Selected organ weight change values

     Dose group (mg/m3)
 Parameter  Sex  0.0  2.3  5.1  12.0
 Thymus (g)  M  0.51  0.41*  0.52*  0.48
 Thymus/body weight %x10  M  1.70  1.39*  1.75  1.63
 Thymus/brain weight %x10  M  2.87  2.22*  2.83  2.67
 Ovary/bodyweight %x10  F  7.18  6.28  5.96*  6.80

Applicant's summary and conclusion

Under the conditions of the test, the test material had no toxic effect at 2.3, 5.1 and 12 mg/m³. The NOEL was determined to be 12 mg/m³.
Executive summary:

In a GLP compliant repeat dose (inhalation) study conducted in line with standardised guideline EU Method B.8, the effects of the repeat dose of the test material in rats was determined.

Under the conditions of the test, no toxic effect was seen at 2.3, 5.1 and 12 mg/m3 and the NOEL was determined to be 12 mg/m3.