Graphite

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-09 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

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted on Sept 7, 2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate attached to full study report.

Limit test:

no

Test material

Test material information

1

Reference substance name:

Graphite

EC Number:

231-955-3

EC Name:

Graphite

Cas Number:

7782-42-5

Molecular formula:

C

IUPAC Name:

carbon

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Strain Wistar Crl:(WI)WU
- Age at study initiation: 8 weeks
- Weight at study initiation: males ca. 250g, females ca. 175g
- 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: 2-3 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

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) = 2.31µm/2.04
Mid dose (MMAD/GSD) = 2.36µm/2.11
High doese (MMAD/GSD) = 2.44µm/2.22

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:
8 mg/m3 (low), 24 mg/m3 (mid), 72 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 2.0), in the low, mid and high dose groups depositited masses of approx. 0.3, 0.7 and 1.8 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 2.0), in the low, mid and high dose groups depositited masses of approx. 0.3, 0.7 and 1.8 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:

no effects observed

Description (incidence and severity):

See section "Details on results"

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See section "Details on results"

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
- One rat was found dead (before a high body weight loss had been measured)
- Upon necropsy, a highly dilatated left kidney space and ureter were observed, with the right kidney being highly enlarged and purative.
- This severe kidney finding is incidentally observed in this rat strain and probably caused by a bacterial infection.
- All other 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 not observed in the treatment groups as compared to controls.

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 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 substance-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 or mild adaptive mucous cell hyperplasia could be observed in this region in a few rats from the Graphite high-dose group of the main study.
- In the nasal cavity, the observed changes were mainly 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 Graphite exposure groups), (adaptive) mucous (goblet) cell hyperplasia (Graphite mid- and high-dose group), subepithelial inflammatory cell infiltration (Graphite mid- and high-dose group), respiratory epithelial hyperplasia (Graphite high-dose group) and squamous cell metaplasia (Graphite high-dose group).
- Eosinophilic inclusions were also observed in a female control rat, and are known as common spontaneous age-related
- The increased 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 and mucous cell hyperplasia was observed at this dose level.
- Thus the Graphite mid-dose group is considered to represent the adverse-effect level in the nasal cavity. There was only a weak decrease of the induced nasal cavity lesions in the animals of the recovery study as compared to the main study.
- In the lung, clearly adverse effects such as markedly increased incidence of interstitial mononuclear cell infiltration and interstitial fibrosis, were seen in the Graphite high-dose group.
- However, due to its high incidence in the Graphite mid- and high-dose group, bronchiolo-alveolar hyperplasia of the bronchiolar type (alveolar bronchiolization) is already interpreted as a mild adverse rather than adaptive effect.
- This type of 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.
- In contrast, bronchial/bronchiolar mucous cell hyperplasia seen in the Graphite high-dose group only is considered as an adaptive non-adverse change. With the exception of mucous cell hyperplasia, all induced changes in the lung as well as in the LALN persisted until the end of the recovery study.
- In all other organs, including larynx and nasopharynx, no effects of the Graphite exposure could be observed.

Effect levels

Dose descriptor:

NOAEC

Effect level:

8 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 Findings Upon Sacrifice

-      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 Upon Sacrifice on Day 29 and Day 56

Treatment	Clean Air	Expanded Graphite low	Expanded Graphite mid	Expanded Graphite high
Aerosol concentration (mg/m^3)	-	8	24	72
Group #	1	2	3	4
Number of Rats(M+F each)	5               5	5*              0	5                 0	5           5
Day	29           56	29            56	29              56	29         56
MALES
Within normal limits	4                   5	0                   -	0                   -	0               0
LUNGS
Multiple grey discoloration	0                   0	4                   -	5                    -	5               2
Multiple grey-black discoloration	0                   0	0                   -	0                    -	5               3
LALN
slightly enlarged	0                   0	0                    -	1                    -	3               3
Grey discolored	0                   0	0                    -	5                    -	5               0
Black discolored	0                   0	0                    -	0                    -	0               5
Testes and Epididymides
Left: Slightly decreased in size	1                   0	0                    -	0                    -	0               0
Right testis: Slightly enlarged	-                     -	1                    -	0                    -	0               0
Left testis: Slightly decreased	-                     -	-                     -	1                    -	0               0
FEMALES
Within normal limits	5                   4	0                   -	0                     -	0               0
LUNGS
Multiple grey discoloration	0                   0	5                   -	4                      -	5               1
Multiple grey-black discoloration	0                   0	0              0	0                      -	0               4
LALN
slightly enlarged	0                   0	-               -	0                     -	0               3
Grey discolored	0                   0	0                   -	5                     -	0               0
Black discolored	0                   0	0                   -	0                     -	2               5
Uterus Horns
Slightly cystic enlargements	0                   1	1                   -	3                     -	3               1
Ovaries
Slightly decreased	0 0	0 -	0 -	2 0

* One rat found dead on day 22 (not included in this table) - LALN: lung-associated lymph nodes

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

- One rat which died before the terminal sacrifice date showed a spontaneous bilateral very severe purulent pyelonephritis (= cause of death) associated with inflammation of a ureter, atrophy of the coagulating glands and seminal vesicles, epithelial erosion of the glandular stomach and lymphoid depletion of the thymus and spleen.

- 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, microgranulomas in the liver, basophilic tubules (tubular basophilia) in the kidneys and subepithelial mononuclear cell infiltration in the larynx.

- 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 Graphite exposure.

- Common spontaneous findings were basophilic tubules (tubular basophilia) in the kidneys (males only) and microgranulomas in the liver occurring at incidences of up to 4/5 rats per group.

Applicant's summary and conclusion

Conclusions:

Based on this 28-day nose-only inhalation study an NOAEL of 8 mg/m3 was derived based on the histopathological examination of the respiratory tract. Expanded 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 was conducted to assess and evaluate potential toxic effects of respirable fractions of Expanded Graphite Powder

-Thirty male and 30 female Wistar rats [strain Crl:WI(WU)] were used for this study and allocated to 4 groups each: Clean Air Control, Expanded Graphite Powder low (8 mg/m³), Expanded Graphite Powder mid (24 mg/m³), and Expanded Graphite Powder high (72 mg/m³). The target aerosol concentrations were achieved satisfactorily, i.e. to 103% - 102% - 99%, respectively.

- One rat was found dead (death due to a severe kidney finding that is incidentally observed in this rat strain).

- All other male and female 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 any 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.

- Absolute and relative lung weights were statistically significantly increased in the high dose group (males) or the mid and high dose group (females). After a 28-day recovery these values were still significantly increased.

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

- The histopathological examination in the nasal cavity showed as dose-dependent changes eosinophilic hyaline cytoplasmic inclusions within respiratory epithelial cells (all Expanded Graphite groups), (adaptive) mucous (goblet) cell hyperplasia (Graphite mid- and high-dose group), subepithelial inflammatory cell infiltration (Graphite mid- and high-dose group), respiratory epithelial hyperplasia (Graphite high-dose group) and squamous cell metaplasia (Graphite high-dose group). Thus 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 high-dose group.

- Based on this 28-day nose-only inhalation study an NOAEL of 8 mg/m3 was derived based on the histopathological examination of the respiratory tract. Expanded Graphite Powder showed effects that were to be expected for a poorly soluble dust with low toxicity.