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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:
2010-04-21 to 2010-08-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP study according to OECD technical guidelines

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
adopted on Sept 7, 2009
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate attached to full study report.
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Strain Wistar Crl:(WI)WU
- Age at study initiation: 11-12 weeks
- Weight at study initiation: males ca 320g, females ca. 190g
- Housing: Makrolon, type III cages, two rats of the same sex and dose group per cage; cages and absorbing softwood bedding material were changed twice a week or more often if necessary
- Diet (e.g. ad libitum): Ssniff V1534, Ssniff Spezialdiäten, Soest, Germany, ad libitum
- Water (e.g. ad libitum): Tap water from Hannover city water supplier, ad libitum
- Acclimation period: 5-6 weeks (to be trained to be accustomed to nose-only tubes)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12
- Air exchange rate: min 10-fold air change per hour

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Low dose (MMAD/GSD) = 2.16µm/1.63
Mid dose (MMAD/GSD) = 2.33µm/1.59
High dose (MMAD/GSD) = 2.34µm/1.66
Details on inhalation exposure:
- Generation of particulate aerosols by dispersing the dry powder
- Dispersion is achieved by a feeding system and a high-pressure, high-velocity pressurized air dispersion nozzle developed by Fraunhofer ITEM.
- An MMAD in the range recommended by the guideline, i.e. approx. 1-3 µm will be generated.
- For each nose-only exposure unit, the aerosol will be generated by a high-pressure pneumatic disperser. The disperser will be fed with the test item under computerized control, i.e. with a feedback loop to the actual aerosol concentrations measured by an aerosol photometer.
- The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant.
- The ratio between photometer signal and concentration will be determined throughout the study by comparing to gravimetric concentrations.
- Filter samples are taken at a free port that is not used for animal exposure. The frequency of filter sampling will be chosen as needed to assure a stable aerosol generation.
- In this system, aerosols will be supplied to each rat individually, and exhaled air is immediately exhausted.
- The airflow to each rat will be approximately 1 l/min which is calculated to be laminar. Therefore measurement of the oxygen concentration is not necessary.
- Prior to the 28-day exposure of rats, technical trials to adjust particle size distributions and exposure levels will be conducted.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please also see details on inhalation exposure.
- Air flow, temperature and relative humidity will be measured continuously and recorded by 20-minute means.
- The limits will be set at 22° C + 2° C for temperature and 55 % + 15 % for relative humidity.
- Additionally, the MMAD will be determined at least two times for each exposure unit (3 units) by a cascade impactor
- The airflow, the temperature and the relative humidity will be monitored continuously and recorded as 10 min mean values.
Duration of treatment / exposure:
6hrs per day, 5 consecutive days per week, 4 weeks
Frequency of treatment:
6hrs per day, 5 consecutive days per week, 4 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
12 mg/m3 (low), 36 mg/m3 (mid), 108 mg/m3 (high)
Basis:
nominal conc.
No. of animals per sex per dose:
28d exposure: 5 males / 5 females per dose
28d recovery in control and high dose, with additional 5 males / 5 females per dose group
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on dose range finding study conducted prior to this study (Fraunhofer ITEM Study-No: 09G0548)
- According to the MPDD Model (v 2.11), in the low, mid and high dose groups depositited masses of approx. 0.45, 1.0 and 2.7 mg/lung can be expected for an aerosol with an MMAD of approx. 2.5µm (3.3% deposition rate)
- Based on this a particle overload situation is induced in the high dose group
- Rationale for selecting satellite groups: To demonstrate reversibility of effects
- Post-exposure recovery period in satellite groups: 28d
Positive control:
- According to the MPDD Model (v 2.11), in the low, mid and high dose groups depositited masses of approx. 0.45, 1.0 and 2.7 mg/lung can be expected for an aerosol with an MMAD of approx. 2.5µm (3.3% deposition rate)
- Based on this a particle overload situation is induced in the high dose group

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (with the exception of weekends and public holidays: once daily), i.e. before and after exposure
- Cage side observations checked in table "Scheduled Investigations in the 28d-study" (Section: Any other information on materials and methods incl. tables) were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Once a week: careful examination for abnormalities concerning observation of the general condition, fur, grooming activity and visible mucous membranes

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week to the nearest 0.1g throughout the study for all animals

FOOD CONSUMPTION:
- Food consumption will be recorded weekly during the study period, including post-exposure observation period

GROSS PATHOLOGY/NECROPSY - ORGAN WEIGHT
- All animals will be subjected to a complete necropsy including careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
- For anesthesia an overdose of carbon dioxide will be used.
- The abdominal cavity will be opened and the diaphragm will be cut carefully allowing the lungs to collapse.
- Heart, esophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN) will be removed from the lung convolution.
- The lung will be inflated under a pressure of about 20 cm water with formalin and will be fixed by immersion for a minimum of 2 hours, and used for histopathology.
- Thereafter the weight of the lower part of the trachea will be recorded and the weight of the lung will be calculated.
- The following organs will be trimmed and wet weights will be recorded: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, and heart.
- All tissues listed in OECD Guideline no. 412, table 2 will be prepared for histopathology.


HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- The hematological and clinico-chemical investigations will be done after the end of exposure following a 16-hour fasting period
- Tap water ad libitum
- 5m/5f animals per dose group; see table "Scheduled Investigations in the 28d-study" (Section: Any other information on materials and methods incl. tables).
- Blood will be taken under slight halothane anesthesia by puncture of the retroorbital plexus.
- Parameters checked in "Hematology and Clinical Chemistry" (Section: Any other information on materials and methods incl. tables) will be examined

URINALYSIS: Yes
- Appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, and leukocytes will be measured semi-quantitatively (5m/5f animals per dose group; see table "Scheduled Investigations in the 28d-study" (Section: Any other information on materials and methods incl. tables).

Sacrifice and pathology:
HISTOPATHOLOGY
- In 5m/5f animals per group after end of exposure.
- For the recovery group animals, all tissues will be preserved but only those showing changes on day 29 will be examined histopathologically.
- full histopathology on the respiratory tract and other organs and tissues, as listed in OECD 412 of all animals in the clean air control group and the Expanded Graphite Powder high dose group and all animals that died or were killed during the study.
- histopathology of lung lobes, including bronchi and the lung-associated lymph nodes (LALN), trachea, larynx, pharynx and the nasal cavities in all animals of all groups.
- Lungs will be fixed in buffered formalin (10%), embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E).
- A special stain will be applied for diagnosis of fibrotic changes: Masson trichrome.
Statistics:
- Differences between groups will be considered statistically significant at p < 0.05.
- Data will be analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups will be compared with the means of the control groups using Dunnett's test.
- The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test by the P.L.A.C.E.S. system.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
(See section "Details on results")
Mortality:
mortality observed, treatment-related
Description (incidence):
(See section "Details on results")
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
(See section "Details on results")
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):
(See section "Details on results")
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
Description (incidence and severity):
(See section "Details on results")
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(See section "Details on results")
Histopathological findings: neoplastic:
no effects observed
Details on results:
SURVIVAL OF ANIMALS AND CLINICAL OBSERVATIONS
- All animals survived the study and, generally, tolerated well the exposure at all concentrations.
- The only relevant clinical observation was an increased rate of eye irritation in some rats of the high dose groups.
- Eye lids appeared slightly red-brownish discoloured. This effect is considered as particle-related, however, it disappeared until the next morning.
- The macroscopical observations made upon sacrifice of animals reflect some test item-related effects such as the test/reference item deposition in the deep lung areas (discoloration), however, severe test item-related adverse effects were not observed macroscopically.

BODY WEIGHT DATA
- Statistically significant changes were observed in the male treatment group during the recovery phase (not in the female groups).
- During the exposure period no statistical significance was found.
- Therefore, this finding is considered as incidental due to a higher body weight in the control group accompanied by a small standard deviation.
- A treatment-related effect cannot be derived from this data.

FOOD CONSUMPTION
- Upon necropsy, test item- or dose-related macroscopical findings were not observed.
- Statistically significant values are considered as incidental findings.

HEMATOLOGY, CLINICAL CHEMISTRY AND URINALYSIS
- Overall, hematological and clinical chemistry data were found in the ranges expected for the species, strain, sex and age.
- Therefore, those findings, albeit significantly different from controls, are considered to represent incidental findings.

HEMATOLOGY
- For red blood cells, hemoglobin and hematocrit statistically significant decreases were observed in the male groups and partially in the female groups.
- Although these are considered as test-item related alterations the alterations are very slight and the values are still in the range usually observed for rats of this species, strain, sex and age (historical data).
- No treatment related adverse effects were detected in the other hematology parameters.

CLINICAL CHEMISTRY
- All values are in the range expected for this species, strain, sex and age.
- Significant differences marked in the tables are considered to be due to biological variance.

URINALYSIS
- All values are in the range expected for this species, strain, sex and age.
- Statistically significant increases were not observed.

GROSS PATHOLOGY/NECROPSY – ORGAN WEIGHTS
- Upon necropsy, test item- or dose-related macroscopical findings were not observed.
- The absolute and relative organ wet weights did show statistically significant changes as compared to controls only in the target organ lungs, i.e. absolute and relative lung weights (males: high dose groups only; females: mid and high dose group).

HISTOPATHOLOGY
- Test particle-related findings were observed in the nasal cavity, trachea, lung and lung-associated lymph nodes (LALN).
- Deposition of particle-laden macrophages was observed in lung, trachea and LALN.
- In the lung, the majority of the particle-laden macrophages were located within the alveoli with a minor portion lodged within the interstitium (perivascular, peribronchiolar and intraseptal).
- At the end of the recovery study, the interstitial portion of particle-laden macrophages was more prominent than after the end of the main study indicating a time-dependent translocation of particles from the alveoli into the interstitium. This is also reflected by a higher burden of particle-laden macrophages in the lung-associated lymph nodes in the recovery animals.
- In the trachea, it was difficult to distinguish whether the subepithelial particle deposits at the carina of the bifurcation were cell-associated or lying freely within the interstitium.
- Although this site is known as a common site for lesions, presumably because this area receives a higher dose from direct aerosol impaction, only minimal adaptive mucous cell hyperplasia could be observed in this region in a single rat from the Graphite mid-dose group.
- In the nasal cavity, the observed changes were all located in the anterio-ventral compartment which is mostly exposed to the airflow.
- Dose-dependent changes were eosinophilic hyaline cytoplasmic inclusions within respiratory epithelial cells (all exposure groups), (adaptive) mucous (goblet) cell hyperplasia (all exposure dose groups) and subepithelial inflammatory cell infiltration (mid- and high-dose group).
- Eosinophilic cytoplasmic inclusions are known as common spontaneous age-related change.
- The significant incidence of eosinophilic globules in the Graphite low-dose group of the present study is interpreted as an adaptive rather than adverse effect, because no association with inflammatory cell infiltration was observed at this dose level.
- In contrast to another study with Expanded Graphite, no respiratory epithelial hyperplasia and no squamous cell metaplasia could be observed in the nasal cavities of the present study.
- In addition, the severity scores for the eosinophilic inclusions were minimally lower than in the previous study.
- The mid-dose group is considered to represent the adverse-effect level in the nasal cavity.
- The same is true for the lung where adverse effects such as significantly increased incidence of interstitial mononuclear cell infiltration, interstitial fibrosis and bronchiolo-alveolar hyperplasia of the bronchiolar type (alveolar bronchiolization) were seen in the mid- and high-dose group.
- The bronchiolar type of bronchiolo-alveolar hyperplasia is considered to be non-preneoplastic and to represent an “attempt of the lung” to facilitate a more efficient removal of inhaled materials via the mucociliary escalator by extension of bronchiolar epithelium into the alveolar ducts.
- Although in contrast to the previous study, bronchial/bronchiolar mucous cell hyperplasia in the lungs was not observed, the adverse lung effects were slightly more pronounced in the present study than in the previous one, probably due to the higher dosage of Graphite used in the present study.
- Although some of the exposure-related findings slightly decreased in incidence and severity like mucous cell hyperplasia in the nasal cavity, they persisted until the end of the recovery time.
- In all other organs, including larynx and nasopharynx, no effects of the test item exposure could be observed.

Effect levels

Dose descriptor:
NOAEC
Effect level:
12 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects in the respiratory tract

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

-Macroscopical observations made upon sacrifice of animals are summarized below (lungs, lung-associated lymph nodes and other organs have been under inspection).

-They reflect some test item-related effects such as the test/reference item deposition in the deep lung areas (discoloration), however, severe test item-related adverse effects were not observed macroscopically.

TABLE: Macroscopical Findings at Sacrifice on Day 31 and Day 58

 

Treatment

 

Clean Air

 

Synthetic

Graphite

low

 

Synthetic

Graphite

mid

 

Synthetic Graphite

high

Aerosol concentration (mg/m3)

-

12

36

108

Group #

1

2

3

4

Number of Rats (M+F each)

5               5   

5               0   

5               0   

5           5   

Day

31           58 

31            58

31            58

31        58

MALES

Within normal limits

2               5      

0                 -      

0                  -      

0           0      

LUNGS

Multiple grey-black discoloured areas

0               0  

5                 -

5                  -

5           5   

LALN

Slight enlargement

0               0  

0                - 

0                 -  

1           0  

Grey-black discolouration

0               0  

0                 -   

2                 -     

5           5  

Testes and Epididymides

Slight reduction in size

3               0  

1                 -  

2                  -

2           0  

 

FEMALES

Within normal limits

4               4      

0                -      

0                 -     

0           0      

LUNGS

Multiple grey-black discoloured areas

0               0  

5                -      

5                 -     

5           5    

LALN

Slight enlargement

0               0  

0                -

0                 -  

3           0

Grey-black discolouration

0           0  

0                -   

5                 -     

5           5  

Kidneys

Yellow-brownish raised area

1           0  

0                -

0                 -  

0           0  

Unilateral cyste

0           1  

0                -

0                 -  

1           0  

Sligh unilateral dilatation

0           0  

1                -

0                  -  

0           0  

Uterus Horns

Slightly cystic enlargement

0       0  

4              -  

1            -  

1     0  

LALN: lung-associated lymph nodes

Histopathology: Non-Item Substance-Related Noteworthy Findings in other Organs

- Common spontaneous findings in groups 1 and 4 from which all protocol organs were examined included estrus cycle-dependent luminal dilatation of the uterus, degeneration of the seminiferous tubules in the testis associated with aspermia, oligospermia and atrophy of the epididymides, myocardial degeneration and mononuclear cell infiltration of the heart, microgranulomas in the liver, basophilic tubules (tubular basophilia), tubular dilatation and transitional cell hyperplasia in the kidneys and epithelial hyperplasia of the thymus.
- These findings occurred at incidences between 2/5 and 5/5 per sex and group and all were considered to be spontaneous and unrelated to Synthetic Graphite exposure.
- Common spontaneous findings were basophilic tubules (tubular basophilia) in the kidneys, microgranulomas and perivascular mononuclear cell infiltration in the liver, degeneration of the seminiferous tubules in the testis and epithelial hyperplasia in the thymus.
- All these findings occurred at incidences between 2/5 and 4/5 per sex and group.

Applicant's summary and conclusion

Conclusions:
Based on this 28-day nose-only inhalation study an NOAEL of 12 mg/m3 was derived based on the histopathological examination of the respiratory tract. Synthetic Graphite Powder showed effects that were to be expected for a poorly soluble dust with low toxicity.
Executive summary:

-  A 28-day nose-only inhalation in the rat according to Commission Regulation (EC) No 440/2008, Part B.8. and OECD Guideline No. 412 to evaluate potential toxic effects of Synthetic Graphite Powder

-  Thirty male and 30 femaleWistar rats [strain Crl:WI(WU)] were used for this study and allocated to 4 groups each: Clean Air Control, Synthetic Graphite Powder low (12 mg/m3), Synthetic Graphite Powder mid (36 mg/m3), and Synthetic Graphite Powder high (108 mg/m3). The target aerosol concentrations of 12, 36 and 108 mg Synthetic Graphite Powder/m3were achieved satisfactorily, i.e. to 103% - 101% - 101%, respectively.

-  All test animals survived treatment and were euthanized at scheduled dates.

-  Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.

-  Body weight development did not show treatment-related statistically significant changes as compared to concurrent controls.

-  Food consumption did not show treatment-related significant changes as compared to concurrent controls. Statistically significant values are considered as incidental findings.

-  Hematology and clinical chemistry data were all in the ranges expected for the species, strain, sex and age. Some statistically significant data are considered as incidental findings.

-  Wet lung weights were statistically significantly increased dose-dependently in the mid and high dose groups of both sexes (absolute data: males, in addition, also in the low dose group - relative data: females, in addition, also in the low dose group). After a 28-day recovery these values were still significantly increased.

-  All other organ weights did not show any treatment-related statistically significant changes as compared to concurrent controls.

-  The histopathological examination in thenasal cavityshowed as dose-dependent changes eosinophilic hyaline cytoplasmic inclusions within respiratory epithelial cells (all Synthetic Graphite groups),(adaptive) mucous (goblet) cell hyperplasia (Graphite low, mid- and high-dose group) and subepithelial inflammatory cell infiltration (Graphite mid- and high-dose group). Overall, the Graphite mid-dose group is considered to represent the adverse-effect level in the nasal cavity.

-  In the lung, clearly adverse effects such as markedly increased incidence of interstitial mononuclear cell infiltration and interstitial fibrosis were seen in the Graphite mid and high dose group.

-  In larynx and nasopharynx and all other organs, no treatment-related effects were observed.

- Based on this 28-day nose-only inhalation study an NOAEL of 12 mg/m3 was derived based on the histopathological examination of the respiratory tract. Synthetic Graphite Powder showed effects that were to be expected for a poorly soluble dust with low toxicity.