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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: RccHanTM: WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Age at study initiation: 9 weeks
- Weight at study initiation:
Males: 220.6 to 240.3 g (mean 229.7 g)
Females: 138.0 to 169.9 g (mean 157.6 g)
- Housing: In groups of five in Makrolon type- 4 cages with wire mesh tops and standard softwood bedding
- Diet: Pelleted standard Harlan Teklad 2914C (batch nos. 53/13 and 10/14) rat maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum)
- Water: Community tap-water from Itingen was available ad libitum in water bottles
- Acclimation period: Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2, and 4 hours, respectively

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: The particle size determination was performed gravimetrically and chemically three times for groups 2 to 4.
The cumulative particle size distribution of the test atmosphere was determined using a modified Mercer seven stage cascade impactor (Model LP 2.5/0.15, Hauke, Gmunden / Austria). The test atmosphere was impacted at each stage onto aluminium slips weighed before and after sampling.

The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the gravimetric results from the impactor, using Microsoft Excel® software (Microsoft Corporation / USA). Additionally, the particle size distribution of the test item in the generated atmosphere was measured by chemically analyzing of the test item deposited on each stage of the cascade impactor. These analyses were determined by Harlan Laboratories Ltd, using an HPLC method.

Gravimetric Determination of Particle Size Distribution

Group Mean MMAD [µm] Mean GSD Number of Determinations Mean Percentage of Particles <3 µm
2 1.64 2.28 3 76.8%
3 2.46 2.59 3 58.3%
4 3.16 2.83 3 48.0%

Chemical Determination of Particle Size Distribution

Group Mean MMAD [µm] Mean GSD Number of Determinations Mean Percentage of Particles <3 µm
2 1.78 2.39 3 72.6%
3 2.26 2.42 3 62.6%
4 2.84 2.57 3 52.3%
Details on inhalation exposure:
Inhalation Exposure System:
Inhalation exposure was performed using a flow-past system. Ports for animal exposure were positioned radially around the nose-only, flow-past exposure chamber on several different levels. The animals were confined separately in restraining tubes. The aerosol was discharged constantly through the exposure system and exhausted using a tubing/filter system.
The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 1 L/min, which is sufficient to minimize re-breathing of the test aerosol as it is more than twice the respiratory minute volume of a rat.

Test Item Generation:
The test item was ground in a ball mill for at least four hours and sieved afterwards.
For groups 3 and 4 a dust aerosol was generated from the test item using a SAG 410 Solid Aerosol Generator (Topas GmbH, Dresden / Germany) connected to a cyclone. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. An air-vac system was used for test item generation for group 2 by splitting of a fraction of the aerosol generated for group 3 and dilution with compressed, filtered, dried air to achieve the concentrations required for this study.

Exposure System Monitoring:
Aerosol concentration, particle size distribution, relative humidity, temperature and oxygen concentration were measured on test aerosol samples taken at a representative exposure port.
All airflow rates (including those for concentration and particle size measurements) were determined using calibrated gas meters and pressure gauges or flow meters.

Temperature, relative humidity and oxygen concentration during exposure were considered to be satisfactory for this type of study.

Group Temperature [°C] Relative Humidity [%] Oxygen Concentration [%]
1 22.4 ± 0.2 (n = 21) 8.6 ± 1.7 (n = 21) 20.7 ± 0.0 (n = 21)
2 21.8 ± 0.1 (n = 21) 15.9 ± 2.1 (n = 21) 20.7 ± 0.0 (n = 21)
3 21.7 ± 0.1 (n = 21) 34.6 ± 2.8 (n = 21) 20.7 ± 0.0 (n = 21)
4 21.8 ± 0.2 (n = 21) 32.9 ± 2.6 (n = 21) 20.7 ± 0.0 (n = 21)

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gravimetric Determination of Aerosol Concentration:
The gravimetric aerosol concentration was determined at least six times during each exposure for groups 2 to 4.
Test atmosphere samples were collected on Durapore Millipore HVLP filters using a stainless steel filter sampling device (Gelman Science Inc., Ann Arbor, Michigan / USA). Sampling flow was 1.0 L/min. The duration of sampling was sufficient to ensure reliable results. The filters were weighed before and immediately after sampling using a calibrated balance (Mettler AG, Volketswil / Switzerland). The aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.

Chemical Determination of Aerosol Concentration:
The actual aerosol concentration in terms of test item was determined twice weekly for groups 2 to 4. On each occasion one or two samples collected for gravimetric analysis were transferred into appropriate vials, sent at ambient temperature to the scientist responsible for formulation analysis (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and measured directly or stored at 2 - 8 °C until analysis. Analysis of the test item were performed using an HPLC method.
Duration of treatment / exposure:
28 days with 20 exposures
Frequency of treatment:
6 hours per day and 5 days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.2, 1.0 and 2.0 mg/L air
Basis:
other: Target conc.
Remarks:
Doses / Concentrations:
0.209 ± 0.024, 1.01 ± 0.074 and 2.01 ± 0.148 mg/L
Basis:
other: Gravimetrically Determined Aerosol Conc.
Remarks:
Doses / Concentrations:
0.199 ± 0.045, 1.12 ± 0.121 and 2.06 ± 0.403
Basis:
other: Chemically Determined Aerosol Conc.
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:Twice daily during acclimatization and treatment.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly (each individual animal) during acclimatization and treatment.

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

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Acclimatization and Week 4 of treatment

- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 Weeks
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table below

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 Weeks
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table below

URINALYSIS: Yes
- Time schedule for collection of urine: After 4 Weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table below

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHT: Yes (see table below)

HISTOPATHOLOGY: Yes (see table below)
Statistics:
The following statistical methods were used to analyze food consumption, body weight, ophthalmoscopic examinations, clinical laboratory investigations, macroscopic findings, organ weights and ratios:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test

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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
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
All animals survived the scheduled treatment period.
There were no clinical signs in any animal during acclimatization or treatment period.

BODY WEIGHT AND WEIGHT GAIN
A very slight body weight loss was seen in males and females at 1.0 and 2.0 mg/L air (1.6 - 3.1%) and females at 0.2 mg/L air (0.6%) during the first four day of treatment. Thereafter normal body weight development was seen in all females and males at 2.0 mg/L air. Males at 1.0 mg/L air showed a statistically significant decrease in body weight gain from day 15 until the end of the study, but mean body weight during this period was similar to as control animals. As no similar findings were recorded at 2.0 mg/L air this was considered to be incidental.
No other differences in body weight were recorded in any group.

FOOD CONSUMPTION
There were no changes in food consumption in any group.

OPHTHALMOSCOPIC EXAMINATION
There were no findings in ophthalmoscopic examination that were considered to be related to treatment with the test item. Corneal opacity was seen in two animals at 0.2 mg/L air, but this was considered to be incidental as it is often seen in rats.

HAEMATOLOGY
There were no changes in heamatology parameters that were considered to be related to treatment. Some differences achieved statistical significance (e.g. hemoglobin in males at 1.0 and 2.0 mg/L air, highly mature reticulocytes in males at 2.0 mg/L air, low and mid mature reticulocytes in females at 0.2 mg/L air, neutrophiles in females at 0.2 and 1.0 mg/L air) but these were within the historical control data or did not show any relationship to aerosol concentrations and were, therefore, considered to be incidental.

CLINICAL CHEMISTRY
Statistically significant decrease in total bilirubin was recorded in females at 1.0 and 2.0 mg/L air (0.87 and 0.86 vs. 2.58 µmol/L), however a decrease of bilirubin level is not considered as an adverse effect. In addition creatinine was statistically significantly increased in males at 1.0 and 2.0 mg/L air due to a low value for the control group compared to the historical data.

There were no other changes in clinical biochemistry parameters that were considered to be related to treatment. Some differences achieved statistical significance (e.g. urea in males at 1.0 mg/L air and females at 2.0 mg/L air, sodium in males at 2.0 mg/L air, potassium in females at 0.2 mg/L air, phosphorus in females at 0.2 mg/L air) but these were within the historical control data or did not show any relationship to aerosol concentrations and were, therefore, considered incidental.

URINALYSIS
There were no changes in urinalysis parameters that were considered to be related to treatment. PH value was statistically significantly decreased in females at 0.2 air and 2.0 mg/L air but this was within the historical control data and was, therefore, considered incidental.

ORGAN WEIGHTS
Statistically significant increase in absolute and relative lung weight was seen in animals receiving the test item. This effect is related to the load of the lungs with the test item and the subsequent cellular infiltration. This effect is not considered as adverse.

In addition statistically significant increase in absolute kidney weight was recorded in females at 1.0 and 2.0 mg/L air (10 and 13%, respectively). Relative kidney weight was increased in females at 2.0 mg/L air (15%). Relative liver (to body weight) and heart (to brain weight) weight were statistically significantly increased in females at 2.0 mg/L air (11 and 16%, respectively). As it was recorded in females only and no correlation with histopathological or biochemistry findings were present this was considered to be of no toxicological significance.

GROSS PATHOLOGY
Gray white discoloration and enlargement of the bronchial lymph nodes was seen in all animals treated with the test item. Gray white discoloration was also seen in the lungs of all females exposed to the test item and all males at 1.0 and 2.0 mg/L air. This finding was also observed in one male at 0.2 mg/L air. In addition an increased incidence of reduced size of prostate (4/5 vs. 1/5 in controls) and seminal vesicles (2/5 vs. 1/5 in controls) was seen in males of the high dose group.

All other findings noted are background findings commonly seen in animals of this age and strain in studies of this type.

HISTOPATHOLOGY: NON-NEOPLASTIC
Lungs and bronchial lymph node:
In the lungs, moderate to marked alveolar macrophages/eosinophilic material was observed from 0.2 mg/L air in males and females with an overall dose related severity reaching plateau at 1.0 mg/L air (see table 1). The changes were characterized by intra-alveolar aggregation of macrophages with foamy cytoplasm and accumulation of finely granular material (most probably the test item). Alveolar macrophages/eosinophilic material was the histological correlate of the increased lung weights and gray white discoloration recorded at necropsy. Minimal to slight infiltration of granulocytes (neutrophils) was also present in the alveoli, admixed with the eosinophilic material and foamy macrophages. The intra-alveolar findings were associated with minimal to slight perivascular infiltration of mononuclear inflammatory cells, mainly lymphocytes with lesser number of macrophages and plasma cells.

In the bronchial lymph node, minimal to moderate foamy macrophage infiltration and increased lymphocytes were observed within the cortex/paracortex from 0.2 mg/L air without clear dose-relationship (see table 1). Foamy macrophage infiltration was characterized by the presence of small foci of enlarged macrophages with a foamy cytoplasm within the cortex and paracortex. Foamy macrophage infiltration and increased lymphocytes were the histological correlates of the gray white discoloration and enlargement recorded at necropsy, respectively.

The mean severity of lungs and bronchial lymph node findings were overall similar in males and females and related to the clearance of the test material from the lungs via the lymphatic system.

Nasal cavity and larynx:
In the nasal cavity, there were minimal focal/multifocal changes within the respiratory and/or olfactory epithelium including squamous metaplasia, hyperplasia, erosion and/or eosinophilic globules from 0.2 mg/L air (see table 2). Although not clearly related to dose, the incidence of most of these findings was higher at 2.0 mg/L air. The squamous metaplasia was characterized by replacement of olfactory, respiratory or transitional epithelium by a few layers of stratified epithelial cells with flattening of the more superficial cells. There was no evidence of cellular atypia or superficial keratinisation. Minimal squamous metaplasia, respiratory epithelium hyperplasia and erosions were observed on the nasal septum, nasoturbinate or maxilloturbinates from cranial sections (levels 1 and/or 2). The epithelial eosinophilic globules were intracytoplasmic, involving most often the respiratory epithelium lining the nasal septum (levels 2 and/or 3).

In the larynx, minimal squamous metaplasia was recorded from 0.2 mg/L air, the incidence being unrelated to dose. The metaplasia involved the transitional epithelium overlying the ventral glands at the base of the epiglottis; there was no evidence of cellular atypia or superficial keratinisation.

There was no test item related findings at the tracheal bifurcation (carina). The effects observed in the nasal cavity and larynx seems to be a non specific response to the deposit of test material in the airways. Considering the low incidence and severity they are not considered as adverse.

Male reproductive organs:
Minimal to moderate bilateral focal/multifocal seminiferous tubule degeneration was observed in the testes with an overall similar mean severity at 0 and 2.0 mg/L Air (see table 3). The changes were characterized by the presence, in some seminiferous tubules, of germ cell disorganization, spermatid giant cells, vacuolation of Sertoli cells, spermatid 19 retention and/or germ cell degeneration. In addition severe seminiferous tubule atrophy was recorded in the testes of 1 of the 5 control males that were found to be reduced in size at necropsy.
At necropsy, the prostate was recorded as reduced in size in 1/5, 1/5, 2/5 and 4/5 rats at 0, 0.2, 1.0 and 2.0 mg/L Air, respectively. The gross finding was correlated histologically with minimal to slight decreased secretory content in some rats (see table 3). Of note, the histological evaluation of the prostate from the affected control rat is pending. No microscopic finding was observed in the seminal vesicles.
Findings in the testis and prostate were considered to be due to stress rather than the test item.

All other findings were considered to be of spontaneous origin, unrelated to the test item. Of note, there was no histological correlate for the increased kidneys weights recorded at necropsy in females.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
> 2.06 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOEC
Sex:
male/female
Basis for effect level:
other: Physiological (not adverse) response to lungs overload by the test item at all concentrations
Remarks on result:
not determinable

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Incidence and severity of test item related microscopic findings in the lungs and bronchial lymph node

 

Males

 

Females

Dose (mg/L Air)

0

0.2

1.0

2.0

 

0

0.2

1.0

2.0

 

Lungs

n

5

1

5

5

 

5

5

5

5

Alveolar foamy macrophage / eosinophilic material

 

 

 

 

 

 

 

 

 

moderate

-

1

-

-

 

-

4

-

-

marked

-

-

5

5

 

-

1

5

5

Mean severity*

0.0

3

4

4

 

0.0

3.2

4

4

Alveolar granulocyte infiltration

 

 

 

 

 

 

 

 

 

minimal

-

1

-

-

 

-

1

-

-

slight

-

-

5

5

 

-

4

5

5

Mean severity

0.0

1

2

2

 

0.0

1.8

2

2

Perivascular mononuclear cell infiltration

 

 

 

 

 

 

 

 

 

minimal

-

-

1

-

 

-

3

1

1

slight

-

1

4

5

 

-

2

4

4

Mean severity

0.0

2

1.8

2

 

0.0

1.4

1.8

1.8

 

Bronchial lymph node

n

5

5

5

5

 

5

5

5

5

Foamy macrophage infiltration

 

 

 

 

 

 

 

 

 

minimal

-

1

-

-

 

-

1

-

1

slight

-

4

1

2

 

-

3

1

2

moderate

-

-

4

3

 

-

1

4

2

Mean severity

0.0

1.8

2.8

2.6

 

0.0

2.0

2.8

2.2

Increased lymphocytes, cortex/paracortex

 

 

 

 

 

 

 

 

 

minimal

-

1

-

-

 

-

-

-

1

slight

-

3

2

1

 

-

2

1

2

moderate

-

1

3

4

 

-

3

4

2

Mean severity

0.0

2.0

2.6

2.8

 

0.0

2.6

2.8

2.2

n: number examined; -  : no animal affected

*: mean severity is ¿ number of animals x severity / number of examined organs in the group

 

Table 2: Incidence and Severity of Test Item Related Microscopic Findings in the Nasal Cavity and Larynx

 

 

Males

 

Females

Dose (mg/L Air)

0

0.2

1.0

2.0

 

0

0.2

1.0

2.0

 

Nasal cavity

n

5

5

5

5

 

3

3

2

3

Squamous metaplasia, focal/multifocal 

 

 

 

 

 

 

 

 

 

minimal

-

1

-

3

 

-

1

-

2

Mean severity*

0.0

0.2

0.0

0.6

 

0.0

0.3

0.0

0.7

Hyperplasia, respiratory epithelium, focus(i)

 

 

 

 

 

 

 

 

 

minimal

-

-

-

1

 

-

1

-

-

Mean severity

0.0

0.0

0.0

0.2

 

0.0

0.3

0.0

0.0

Erosion, respiratory epithelium focal/multifocal

 

 

 

 

 

 

 

 

 

Minimal

-

-

-

2

 

-

-

-

1

Mean severity

0.0

0.0

0.0

0.4

 

0.0

0.0

0.0

0.3

Erosion, olfactory epithelium focal/multifocal

 

 

 

 

 

 

 

 

 

Minimal

-

-

1

-

 

-

-

-

1

Mean severity

0.0

0.0

0.2

0.0

 

0.0

0.0

0.0

0.3

Eosinophilic globules, epithelial cells

 

 

 

 

 

 

 

 

 

Minimal

-

1

5

2

 

-

2

2

1

Slight

-

1

-

-

 

-

-

-

1

Mean severity

0.0

0.6

1.0

0.6

 

0.0

0.7

0.7

1.0

 

Larynx

n

4

5

5

5

 

5

5

4

5

Squamous metaplasia

 

 

 

 

 

 

 

 

 

minimal

-

4

4

4

 

-

4

4

4

Mean severity

0.0

0.8

0.8

0.8

 

0.0

0.8

1.0

0.8

n: number examined; -  : no animal affected

*: mean severity is ¿ number of animals x severity / number of examined organs in the group

 

Table 3: Incidence and severity of noteworthy microscopic findings in testes and prostate

Dose (mg/L Air)

0

0.2

1.0

2.0

 

Testes

n

5

0

0

5

Seminiferous tubule degeneration, focal/multifocal

 

 

 

 

minimal

-

 

 

3

slight

2

 

 

1

moderate

1

 

 

-

Mean severity*

1.4

 

 

1.0

Seminiferous tubule atrophy

 

 

 

 

severe

1

 

 

-

Mean severity

1.0

 

 

0.0

 

Prostate

n

0

1

2

4

Decreased secretory content

 

 

 

 

minimal

 

-

1

2

slight

 

-

-

1

Mean severity

 

0.0

0.2

0.8

n: number examined; -  : no animal affected

*: mean severity is ¿ number of animals x severity / number of examined organs in the group

Applicant's summary and conclusion

Conclusions:
Inhalation exposure to EKKE for 28 days to Wistar rats at aerosol concentrations of 0.2, 1.0 and 2.0 mg/L air resulted in changes of the lung and the bronchial lymph nodes.
Inflammatory changes in the lungs were characterized by the presence, within the alveoli, of macrophages with foamy cytoplasm, granulocytes (neutrophils) and accumulation of finely granular material (the test item). Perivascular infiltration of mononuclear inflammatory cells, mainly lymphocytes, was also observed within the lung parenchyma. The eosinophilic material was considered to be the result of macrophages membrane cell rupture. The changes in the bronchial lymph node were consistent with the drainage of pulmonary foamy macrophages associated with reactive lymphoid hyperplasia within the cortex/paracortex. The increase of macrophages correlates to the lung load with the test item and was considered to be related to pulmonary clearance. In the absence of any degenerative lesion in the lungs, these findings were considered to be a normal physiological response to an insoluble particle and not adverse.
Minimal squamous metaplasia, respiratory epithelium hyperplasia, erosion and/or eosinophilic globules in the nasal cavity and squamous metaplasia in the larynx were observed in all test item groups and indicated a minimal irritant effect of the test item. Although not clearly related to dose, the incidence of most of nasal findings was higher at 2.0 mg/L air. Cytoplasmic eosinophilic globules are frequently observed in inhalation studies (Renne et al., 2009) and are considered as a non specific response to mucosal irritation (Greaves, 2007). Squamous metaplasia in the larynx at the base of the epiglottis is a common reaction to inhaled material and, when of minimal severity, is considered to be a non-adverse adaptative change (Kaufmann et al., 2009).
The high incidence of seminiferous tubule degeneration recorded in the testes from rats of the control and high dose groups was most likely related to the stress associated with immobilization in the restrainer during the inhalation procedure (Lee et al. 1993). The prostate was found to be reduced in size at necropsy in all groups including control group with however an apparent dose-related increased incidence. The gross finding was correlated histologically with decreased secretory content in some rats. Owing to the high incidence of seminiferous tubule degeneration recorded in the testes in control and high dose groups attributed to the experimental procedure and absence of microscopic findings in the seminal vesicles and the lack of relationship between dose and mean severity of prostate microscopic findings, the macroscopic and microscopic prostatic findings were considered to be most likely unrelated to the test item, probably also linked to the experimental procedure.
Based on the results of this study, the no-observed-adverse-effect-concentration (NOAEC) was 2.0 mg/L air, a NOEC could not be determined.
Executive summary:

In a study compliant to the OECD test guideline no. 412, EKKE was administered daily by nose-only, flow-past inhalation to Wistar rats of both sexes at achieved aerosol concentrations of 0.2, 1.12 and 2.06 mg/L air for a period of 28 days. A control group was treated similarly with air, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Clinical signs, food consumption and body weights were recorded periodically during the acclimatization and treatment periods. Clinical laboratory investigations were performed during week 4. At the end of the dosing all animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

The chemical mean mass median aerodynamic diameter (MMAD) was between 1.78 and 2.84µm, showing that the generated aerosols were within the respirable range for rats. Temperature, relative humidity and oxygen were considered to be satisfactory for this type of study.

All animals survived the scheduled treatment period. There were no clinical signs and no effects on food consumption. A very slight body weight loss was seen during the first treatment days in males and females at 1.0 mg/L air and 2.0 mg/L air and females at 0.2 mg/L air. Thereafter no differences in body weight gain were seen that were considered to be related to treatment with the test item. There were no treatment-related or biologically significant changes in ophthalmoscopic findings, hematology, clinical biochemistry and urinalysis. Increase in absolute and relative lung weight was seen in animals receiving the test item and was related to the load of the lungs with the test item and the subsequent cellular infiltration.. In addition increase in kidney weight was recorded in females at 1.0 mg/L air (absolute only) and 2.0 mg/L air (absolute and relative). Relative liver (to body weight) and heart (to brain weight) weight were statistically significantly increased in females at 2.0 mg/L air. As it was recorded in females only and no correlation with histopathological or biochemistry findings were present this was considered to be of no toxicological significance.

Gray white discoloration and enlargement of the bronchial lymph nodes was seen in all animals treated with the test item. Gray white discoloration was also seen in the lungs of all females exposed to the test item and all males at 1.0 mg/L air and 2.0 mg/L air.

Moderate to marked alveolar macrophages/eosinophilic material in the lungs was recorded in all test item groups with a dose related severity. That was associated with minimal to slight alveolar infiltration of granulocytes (neutrophils) and minimal to slight perivascular infiltration of mononuclear inflammatory cells, mainly lymphocytes. Minimal to moderate foamy macrophage infiltration and increased lymphocytes within the cortex/paracortex of bronchial lymph node was observed in all test item groups without clear dose-relationship. Minimal squamous metaplasia in the nasal cavity (focal, unilateral) and larynx (at the base of the epiglottis) was seen at 2.0 mg/L air. The increase of macrophages correlates to the lung load with the test item and was considered to be related to pulmonary clearance. In the absence of any degenerative and inflammatory lesion in the lungs, these findings were considered to be a normal physiological response to an insoluble particle and not adverse.

Based on the results of this study, the no-observed-adverse-effect-concentration (NOAEC) was higher than 2.06 mg/L, a NOEC could not be determined.