Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Mar - 9 Jul 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bundesamt für Sicherheit im Gesundheitswesen, Wien, Austria
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation:
- Fasting period before study: animals were fasted overnight prior to administration.
- Housing: animals were housed in groups of 3 per sex in the dose range finding study. In the main study, animals were housed in groups of 2-3 per cage per sex in Makrolon cages Type IV with wire mesh lids.
- Diet: Ssniff R/M-H maintenance diet for rats and mice (V1534-300, Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, acidified with HCL to pH ≥ 3, ad libitum
- Acclimation period: about 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 (average dose range finding study), 19.8 (average main study)
- Humidity (%): 59.1 (average dose range finding study), 56.6 (average main study)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: ethanol
Remarks on MMAD:
MMAD / GSD: MMAD: 1.11, 1.15 and 1.22 µm (low-, mid- and high-dose)
GSD: 1.68-2.57
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: head nose only exposure unit (TSE-Systems GmbH, Bad Homburg, Germany)
- Method of holding animals in test chamber: animal exposure cage
- Source and rate of air: compressed air supply or compressed air generator
- Method of particle size determination: the size distribution of the aerosol particles was analysed with a cascade impactor (Berner-Impaktor, Type LPI40,06/2, Hauke KG, Gmunden, Austria)

TEST ATMOSPHERE
- Brief description of analytical method used: the aerosol concentration in the breathing zone was determined gravimetically.
- Samples taken from breathing zone: yes

VEHICLE
- Justification for use and choice of vehicle: the original high viscous liquid could not be pressed through the aerosol nozzle. Therefore the test substance was mixed with ethanol to decrease the viscosity. In the high-dose group the concentration was 10%. It was the target to have the same concentration of ethanol in all dose groups and in the control group. Therefore different concentrations of ethanol in the test substance were used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The aerosol concentration in the breathing zone was determined gravimetrically. An accurately measured volume of air from the inhalation devices was sucked through a pre-weighed filter with cotton wool. The filters were dried before and after the sampling by pressing dry air through them. From the weight difference and the volume the actual aerosol concentration was calculated.
Duration of treatment / exposure:
4 weeks (main study)
4 weeks and 2 weeks post-exposure observation period (satellite control and high-dose test group)
Frequency of treatment:
6 h/day, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
1.2 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)
Dose / conc.:
4 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)
Dose / conc.:
11.6 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)
Dose / conc.:
1.3 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)
Dose / conc.:
4.3 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)
Dose / conc.:
12.9 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)
Dose / conc.:
0.006 mg/L air (nominal)
Dose / conc.:
0.02 mg/L air (nominal)
Dose / conc.:
0.06 mg/L air (nominal)
Dose / conc.:
6 mg/m³ air (nominal)
Dose / conc.:
20 mg/m³ air (nominal)
Dose / conc.:
60 mg/m³ air (nominal)
No. of animals per sex per dose:
3 (preliminary study)
5 (main study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose ranges are based on the results of the dose range finding study. In the high-dose group (0.6 mg/L) and mid-dose group (0.19 mg/L) all animals died on the day of administration. Therefore, the dose of 0.06 mg/L, which was the low-dose in the dose range finding study, was chosen as high-dose for the main study. The low dose was set at 10% of the high dose; the mid-dose was interpolated geometrically.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were carefully observed for general signs and the health status before, during and after the exposure; on days without exposure once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice per week

FOOD CONSUMPTION:
- Food consumption per cage in weekly intervals in all animals. Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 (main study groups) and Day 43 (satellite groups)
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes
- How many animals: All animals of main study and satellite groups.
- Parameters checked: red blood cell count (RBC), haemoglobin concentration (Hb), haematrocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean cell volume (MCV), reticulocyte count (% of erythrocytes), white blood cell count (WBC), platelet count, differential white blood cell count (% of the different cell species), prothombin time (Quick) as indicator of blood clotting capacity

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (main study groups) and Day 43 (satellite groups)
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes
- How many animals: All animals of main study and satellite groups.
- Parameters checked: aspartate aminotransferase, alanine aminotrasferase, albumin, alkaline phosphatase, bilirubin, calcium, chloride, cholesterol, creatinine, gamma glytamyl transferase, globulin, glucose, phosphorus, potassium (K+), sodium (NA+), total protein, triglycerides, urea
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Animals were killed and subjected to gross pathological examination immediately after death.
HISTOPATHOLOGY: Yes. Gross lesions, tissue masses or tumours, adrenal glands, brain (including cerebrum, cerebellum and pons), eyes, heart, kidneys, larynx (3 levels, including the base of the epiglottis), liver, lungs (all lobes at one level, including main bronchi), lymph nodes (hilar region of the lungs), nasopharyngeal tissues (4 levels), oesophagus, ovaries, seminal vesicles, spinal cord (cervical, thoracal, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid glands, trachea (longitudinal section through the carna and 1 transverse section), uterus
Statistics:
Analysis of variance followed by the Scheffe-test was used for all data with means and standard deviations with comparison of more than two groups. The t-test was used for all data with means and standard deviations for comparison of two groups only. H-test of Kruskal and Wallis followed by the test of Nemenyi counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled. Chi2-test was used for counted events. Fisher´s exact test was used for counted events, if the Chi2-test was not applicable.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
mid- and high dose males and females and female control group: chromodakryorrhoea; high dose satellite group males and low- and high-dose group females: reduced well-being
Mortality:
mortality observed, treatment-related
Description (incidence):
mid- and high dose males and females and female control group: chromodakryorrhoea; high dose satellite group males and low- and high-dose group females: reduced well-being
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high-dose groups: generally lower body weight gain
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):
high-dose males: increased MCV, high-dose males after recovery: reduced WBC, lymphocytes; mid-dose females: decreased reticulocyte count, non adverse
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
low-dose females: decreased chloride, increased phosphor; high-dose females increased AP and ALT; low-dose females: increased ALT, all females: decreased CHOL during dosing, increased at the end of the recovery period
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):
significant changes in organ weights are summarized in Table 3 under "Any other information on results incl. tables".
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
adaptive responses to the deposits of the test substance and the resulting irritation (squamous metaplasia and epithelium proliferation and submucous acute inflammation at the basis of the epiglottis).
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived until the end of the study period. Chromodakryorrhoe occurred in male animals of the mid and high dose groups. This finding was also observed in females of the mid- and high-dose and control group. The findings may be caused by the stress during the administration procedure.
Signs of reduced well-being were noted in male animals of the high dose satellite group and in females of the low and high dose group. Stress can also be the cause for these findings. The effects were seen during the administration period and were not observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
Body weight and body weight gain were lower in the high dose groups. In the mid dose group an effect was observed only in male animals and only at the end of the administration period (see Table 1 under “Any other information on results incl. tables”).

FOOD CONSUMPTION
No significant effects on food consumption were noted during the study period.

HAEMATOLOGY
Changes in the MCV, WBC and lymphocytes in male animals of the high dose groups were observed. In female animals of the mid-dose group reticulocyte counts were significantly reduced. The effects may be random events, because no other parameters are impaired (see Table 2 under “Any other information on results incl. tables”).

CLINICAL CHEMISTRY
No significant effects on clinical chemistry parameters were observed in male animals.
In females of the low-dose group increased phosphor, decreased cholesterol and increased AST was apparent. In the mid-dose group the cholesterol level was decreased as well. In high-dose females chloride level was decreased and normal at the end of the recovery period. Cholesterol was decreased during the dosing period and increased at the end of the recovery period in high-dose females. AP and ALT were statistically significant increased in the high dose group and normal after the recovery period. The changes of cholesterol in all females dosing groups may be caused by the test substance. However, corresponding damages of the liver were not found in the animals at necropsy, histopathology and in organ weights.
All other parameters and statistically calculated significances do not show a dose response relationship and/or are very small changes and are therefore assumed to be random events without toxicological importance (see Table 2 under “Any other information on results incl. tables”).

ORGAN WEIGHTS
Different significant changes in organs weights in the mid- and high-dose male animals were observed (see Table 3 under “Any other information on results incl. tables”). The changes in testes were also seen in the high-dose satellite group and can therefore be considered as not reversible. Changes in spleen were noted in the high-dose satellite group only. The author assumed that such an effect may be test substance related, and occur only after the end of the administration period.
In female animals relevant changes were noted in the high-dose group only. The changed parameters and statistically calculated significances in female animals do not show a dose-relationship. Therefore, the changes are considered to be random without toxicological importance.
In general the increase in relative organ weights may be associated with the reduced body weight and body weight gain.

GROSS PATHOLOGY
No test substance related changes were observed at the gross examination.

HISTOPATHOLOGY: NON-NEOPLASTIC
The test substance aerosol caused several effects in the respiratory system, in lungs and bronchi. Most effects were adaptive responses to the deposits of the test substance and the resulting irritation (squamous metaplasia and epithelium proliferation and submucous acute inflammation at the basis of the epiglottis). In addition focal early stage of fibrosis was noted at the alveoli walls mainly in the groups treated with the test substance. Due to the high amount of the test substance deposits in the lungs especially in the mid and high dose groups these changes may be explained by overloading the tissue and not necesarrily imply an intrinsic toxicity of the test material. The author considered an intrinsic toxicity as unlikely as the test material is insoluble and the shape of the particles is not fibrous.
Some other findings were considered as unspecific and of no toxicological relevance. However, most findings were observed in the high-dose groups.

OTHER FINDINGS
RESULTS OF THE DOSE RANGE FINDING STUDY
All animals of the mid and high dose group died on Day 1 acutely still during the exposition and/or directly thereafter (aerosol concentration 0.19 mg/L and 0.6 mg/L, respectively). No indication for a test substance related alteration in animals of the low dose group was noted (aerosol concentration 0.06 mg/L)). Body weight was lower in the high dose groups. In the mid dose group an effect was observed only in male animals and only at the end of the administration period. There were no statistically significant changes in the low dose groups in body weight, feed consumption and at necropsy.

AEROSOL CONCENTRATION
The mean actual aerosol concentrations were 0.006, 0.02 and 0.059 mg aerosol per liter air for the low, mid- and high-dose groups. The values were converted to the inhaled amount (dose) using the equation: minute_volume (L/min) = body weight (kg)^0.75 x 0.373 (Guyton´s equation) for the breathing volume of the animals.
The calculation gave doses of 11.6/12.9, 4.0/4.3 and 1.2/1.3 mg/kg/day for the high-, mid- and low-dose groups (males/females).
The inhaled amount of ethanol in every group is expected to be about 1/10 of the test substance amount (1 mg/kg/day in the high-dose group).


Effect levels

open allclose all
Dose descriptor:
NOEC
Effect level:
< 0.006 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects on the respiratory system with indications for a local irritation
Dose descriptor:
NOAEC
Effect level:
> 0.06 mg/L air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: effects on organ weight without corresponding effects in histopathological examination and without dose-relationship

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Body weight (g) presented as mean ± SD (males and females).

Dose Level

Body weight (g) on Days / Males

 

1

12

15

19

22

26

28

33

36

40

42

Control

205 ± 8.73

221 ± 13.3

218 ± 14.1

232 ± 14.0

229 ± 13.2

240 ± 13.0

237 ± 14.6

-

-

-

-

Control satellite

203 ± 3.21

245 ± 2.45

215 ± 2.92

227 ± 2.88

225± 1.82

236 ± 1.95

232 ± 0.84

246 ± 3.11

254 ± 5.39

265 ± 5.89

265 ± 6.06

Low-dose

200 ± 9.18

212 ± 9.42

210 ± 8.5

221 ±10.4

216 ± 7.29

226 ± 7.76

221 ± 8.79

-

-

-

-

Mid-dose

200 ± 6.46

210 ± 5.07

204 ± 4.76

219 ± 4.83

212 ± 4.47

225 ± 4.97

216 ± 5.63*

-

-

-

-

High-dose

200 ± 9.5

206 ± 8.7

200 ± 9.07

209 ± 9.18*

200 ± 8.41*

211 ± 8.94*

202 ± 7.23*

-

-

-

-

High-dose satellite

202 ± 7.96

207 ± 8.83

199 ± 9.36±

212 ± 9.64*

202 ± 8.58*

213 ± 10.8*

203 ± 8.29*

223 ± 10.4*

234 ± 12.1*

247 ± 13.1*

247 ± 16.4

Dose Level

Body weight (g) on Days / Females

 

1

22

26

28

33

36

40

42

Control

140 ± 3.13

149 ± 4.49

153 ± 4.51

152 ± 5.41

-

-

-

-

Control satellite

137 ± 3.16

146 ± 4.1

152 ± 5.59

150 ± 4.87

158 ± 5.5

162 ± 6.38

168 ± 7.8

166 ± 9.13

Low-dose

138 ± 4.27

142 ± 9.15

146 ± 9.2

143 ± 9.02

-

-

-

-

Mid-dose

141 ± 4.82

145± 2.95

151 ± 2.7

147 ± 4.04

-

-

-

-

High-dose

138 ± 4.87

136 ± 5.1*

142 ± 4.3*

136 ± 4.97*

-

-

-

-

High-dose satellite

140 ± 6.19

138 ± 5.85*

141 ± 8.17*

138 ± 5.97*

148 ± 6.91*

153 ± 6.8

164 ± 7.3

162 ± 6.73

*: significant different from control group p < 0.05

Table 2. Mean haematological data and clinical chemistry (only significant changes are given).

Parameter

 

Results of statistical calculation

Males

 

Females

Control

Control satellite

High dose

High dose satellite

Control

Control satellite

Low dose

Mid dose

High dose

High dose satellite

MCV (mean corpuscular volume fL)

47.3 ± 0.95

46.9 ± 0.17

49.3 ± 1.33*

47.1 ± 0.83

-

-

-

-

-

-

WBC (white blood cell count, 1/nL)

7.82 ± 1.73

6.12 ± 1.3

5.66 ± 0.49

4.89 ± 0.49*

-

-

-

-

-

-

Lymphocytes (% of leukocytes or n 1/nL)

5.37 ± 1.28

4.42 ± 1.32

3.56 ± 0.47

3.21 ± 0.24*

-

-

-

-

-

-

Reticulocyte count (%), males

-

-

-

-

3.69 ± 0.36

-

-

2.58 ± 0.33*

-

-

Chlorid (mmol/L)

-

-

-

-

99.9 ± 0.72

99.7 ± 1.19

-

-

98.2 ± 0.77*

100 ± 1.07

Phosphor (mmol/L)

-

--

-

-

2.53 ± 0.23

-

3.09 ± 0.43*

-

-

-

Cholesterol (mmol/L)

-

-

-

-

2.37 ± 0.15

2.34 ± 0.06

1.90 ± 0.26*

1.87 ± 0.27*

1.83 ± 0.14*

2.6 ± 0.2*

AP (alkaline phosphatase, U/L)

-

-

-

-

118 ± 5.34

100 ± 7.6

-

-

138 ± 7.97*

103 ± 4.04

AST (aspartate aminotransferase, U/L)

-

-

-

-

92.2 ± 2.68

91.4 ± 7.47

105 ± 6.88*

-

-

-

ALT (alanin aminotransferase, U/L)

-

-

-

-

61.4 ± 6.43

64.4 ± 2.88

-

-

75.4 ± 7.99*

62.2 ± 1.79

*: significant different from control group p < 0.05

Table 3. Mean organ weights. Only significant changes are given.

Parameter

Results of statistical calculation

Males

Females

Control / Control satellite

Mid dose

High dose

High dose satellite

Control / Control satellite

High dose

High dose satellite

Absolute organ weights (mg)

Testes

2283 ±185

1966 ± 162

2716 ± 123*

2771 ± 42.4 *

2651 ± 204*

-

-

-

Spleen

512 ± 66.7

645 ± 48.4

-

-

531 ± 59.2*

-

-

-

Lung

-

-

-

-

1173 ± 118

926 ± 76.9

-

1104 ±154*

Thymus

291 ± 60.7

313 ± 51.5

186 ± 17.9*

188 ± 17.9*

261 ± 51.9

-

-

-

Organ weight/body weight ratios (mg/g)

Brain

7.86± 0.55

7.42 ± 0.19

5.66 ± 0.37*

9.0 ± 0.23*

7.61 ± 0.44

11.2 ± 0.74

10.4 ± 0.31

12.9 ± 0.46*

10.6 ± 0.46

Lung

-

-

-

-

7.69 ± 0.68

5.57 ± 0.47

-

6.79 ± 0.86*

Heart

3.42 ± 0.43

3.51 ± 0.22

-

3.97 ± 0.23*

3.68 ± 0.35

3.88 ± 0.11

3.38 ± 0.26

4.27 ± 0.18*

3.81 ± 0.25

Testes

9.64 ± 0.43

7.41 ± 0.19

12.6 ± 0.76*

13.7 ± 0.3*

10.8 ± 1.13*

-

-

-

Spleen

2.17 ± 0.29

2.43 ± 0.16

-

-

2.14 ± 0.18*

-

-

-

Kidneys

6.86 ± 0.65

6.65 ± 0.62

-

8.01 ± 0.53*

6.6 ± 0.52

8.08 ± 0.41

6.43 ± 0.35

-

7.28 ± 0.66*

Thymus

1.22 ± 0.18

0.86 ± 0.06*

-

-

-

-

-

Organ weight/brain weight ratios (mg/g)

Heart

-

-

-

-

-

-

-

Spleen

277 ± 41.1

325 ± 23.5

-

-

282 ± 24.5*

-

-

-

Thymus

157 ± 32.7

159 ± 24.9

99.4 ± 8.15*

103 ± 23.9*

139 ± 29.0

-

-

-

Kidneys

-

 

-

-

719 ± 31.3

618 ± 20.1

-

685 ± 53.9*

Testes

1231 ± 92.5

1000 ± 93.8

1456 ± 75.4*

1522 ± 41.8*

1415 ± 47*

-

-

-

*: significant different from control group p < 0.05

Applicant's summary and conclusion

Conclusions:
The used concentration range of the study was chosen due to a range finding test. The aerosol of the test substance caused effects at several sites of the respiratory system at concentrations of 0.006 to 0.060 mg/L with indications for a local irritation. The alterations may be explained by the amounts of inert material deposited within the respiratory system. In the lungs and bronchi the most prominent finding was a focal early stage of fibrosis. Due to the high amount of test substance deposits in the lungs especially in the mid and high dosed groups these changes may be explained by simply overloading the tissue and do not necessarily imply an intrinsic toxicity of the test material. An intrinsic toxicity is unlikely as the test material is insoluble and the shape of the particles is not fibrous.
Alterations in clinical biochemistry were only seen in female animals. Beside this and the increased absolute and relative organ weights of testes in male animals there was no other substantially sex difference in the response to the test substance. Furthermore, the differences did not show a dose relationship and no correlating effects on the respective organ were seen in the histopathological examinations.
The observed effects were at most mild in severity, never became life threatening, but persisted partly until the end of the recovery period. Furthermore, due to the self-warning characteristics (immediate irritant reactions to the test substance if inhaled), high substance concentrations of inhalation may be avoided.
The No-observed-effect-concentration (NOEC) for local irritation of the test material via inhalation was smaller than 0.006 mg/L. There was however no life threatening systemic effect present up to the highest concentration tested. Therefore, the systemic NOAEC for the test material will be > 0.06 mg/L.