[No public or meaningful name is available]

Carcinogenicity

Administrative data

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Nov 2010 - 9 Nov 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Principles of method if other than guideline:

- Principle of test: Repeated doses of test substance were administered to male hamsters by intratracheal instillation to assess the carcinogenic potential of the test item in the respiratory tract and mesothelium during the 2-year breeding period.
- Short description of test conditions: The test item was administered to 12-week-old male hamsters. As a vehicle, 5% DMSO–Eagle’s MEM was used. Terminal necropsy was performed during week 104 after the first dosing.
- Parameters analysed / observed: Animals were observed for clinical signs, weighed, necropsied, and examined histopathologically.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Vapour grown graphitic carbon fibre

Molecular formula:

C

IUPAC Name:

Vapour grown graphitic carbon fibre

Test material form:

solid

Test animals

Species:

hamster, Syrian

Strain:

other: Slc

Details on species / strain selection:

Syrian hamsters have been used in intratracheal instillation studies to assess carcinogenicity in the respiratory tract. Male hamsters show slower body weight gain and lower interindividual variability in body weight than female hamsters.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan SLC, Inc.
- Age at study initiation: 12 weeks
- Housing: Aluminum cages with sheets of woven stainless-steel-wire on the front and at the bottom. Cages and racks were replaced every 2 months. Animals were housed individually.
- Diet: Lab Diet #5002 (PMI Feeds, USA), ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 9 Oct 2010 To: 9 Nov 2012

Administration / exposure

Route of administration:

intratracheal

Type of inhalation exposure (if applicable):

other: forced intratracheal instillation

Vehicle:

other: 5% DMSO–Eagle’s minimal essential medium (MEM)

Details on exposure:

DOSE PREPARATION
High dose: 120 mg test item was weighed with an electronic balance, supplemented with 1.5 mL DMSO, and sonicated for 5 min with an ultrasonic cleaner. The solution was supplemented with 28.5 mL Eagle’s MEM and sonicated for 5 min with an Ultrasonic Disruptor (UD-201; Tomy Seiko, Japan).
Low dose: Solutions were prepared by 10-fold dilution of high dose solution.
The homogeneity was inspected visually.

METHOD OF ADMINISTRATION
Administration by forced intratracheal instillation under anesthetized conditions. Test item was injected into the trachea of animals as if it was sprayed by using a 1-mL disposable syringe attached to an adapter with a processed silicon tube. 0.05 mL of dosing solution was discharged from a syringe with 0.45 mL of air.

METHOD OF ANESTHESIA
Intraperitoneal injection of 0.2 mL Ketalar® 50 (Ketamine 50 mg/mL; Sankyo Lifetech, Japan) and Selactar® 2% (Xylazine 20 mg/mL; Bayer Medical, Japan) in a ratio of 9:1.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

1 administration / week

Post exposure period:

2 years

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.02 other: mg/animal/administration

Remarks:

0.12 mg/animal (total dose), corresponding to 44.4 mg/L air

Doses / concentrations 2

Dose / conc.:

0.2 other: mg/animal/administration

Remarks:

1.2 mg/animal (total dose), corresponding to 444 mg/L air

No. of animals per sex per dose:

49 in low dose group (one animal died from anesthesia during administration)
50 in high dose and control group, each

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a prior instillation-carcinogenicity study, animals receiving a total dose of 12 mg were found to be in a state of lung overload.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed daily for clinical signs until the day of necropsy. Detailed observations were performed once per week.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the first day of the study, at 3 and 6 weeks of dosing, and every 4 weeks thereafter until completion of the study. They were also weighed on the day of terminal necropsy. Dead animals were weighed at the time when they were found dead.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Various organs, especially the lungs, were observed macroscopically for all animals.

HISTOPATHOLOGY: Yes
Lungs and masses observed in the abdominal and thoracic cavities were examined with a light microscope.

Statistics:

Body-weight data were first tested for homogeneity of variance by Bartlett’s test (level of significance, 5%). If homogeneity was found, a one-way analysis of variance was performed (level of significance, 5%). If homogeneity was found in the analysis, Scheffé's multiple comparison test was performed (two-sided significance level, 5% and 1%). If homogeneity was not found, the Kruskal-Wallis test was performed (level of significance, 5%). If a significant difference was detected in the test, the Scheffé-type rank test was performed (two-sided significance level, 5% and 1%).

Tumor incidence was tested by Fisher’s test (one-sided significance level, 5% and 1%). The data obtained from test animals that survived for at least 1.5 years after the first dosing were included in analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Findings such as emaciation were noted in animals that died during the study period.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One animal of the low dose group died from anesthesia on the day of the fourth dosing (day 28 of the study). Survival rate was 31% and 50% at 2 years after the first dosing in the low and high dose group, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant decrease in body weight was found in any of the test item groups compared with the control group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Lungs: black dots, black spots and blackening (all observed in low and high dose)
Thoracic cavity: Hydrothorax (low and high dose) and intrathoracic mass (low dose)
Abdominal cavity: Ascites (low and high dose), intra-abdominal mass (high dose)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance and control groups: Alveolitis, fibrosis of the alveolar walls, and bronchiolar epithelial metaplasia; alveolar proteinosis and squamous metaplasia in the focus of the disease; alveolar and bronchiolar epithelial hyperplasia
Test substance group: deposition of test substance and macrophages laden with the deposited test substances; as well as nodular lesions with fibrosis

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Bronchiolar epithelial metaplasia and hyperplasia. Also noted in the control group and not considered to be precancerous lesions.

A mesothelioma was found in the abdominal cavity of one animal in the high-dose group. Not considered to be induced by the test item.

Details on results:

Macrophages laden with pigment-like components (including a hemosiderin-like component produced by hemorrhage) were observed in a scattered manner in the test item groups as well as the vehicle control group. In addition, the following specific changes were observed in the test item groups. The occurrence and accumulation of macrophages laden with test item and nodular lesions with fibrosis. The severity of these changes was higher in the high dose group than in the low dose group. However, all these differences in severity or level were slight. In addition, test item-laden macrophages were observed in the parabronchial lymphoid tissue in the test item groups.

The following lesions were also noted in all treatment groups including the vehicle control group:
i) Alveolitis as well as fibrosis of the alveolar walls and bronchiolar epithelial metaplasia;
ii) Alveolar proteinosis and squamous metaplasia in the focus of the disease;
iii) Alveolar and bronchiolar epithelial hyperplasia.

Of the above-mentioned changes, alveolitis as well as fibrosis of the alveolar walls and bronchiolar epithelial metaplasia in i) were not obviously higher in the test item groups than in the vehicle control group. Similarly to these lesions, the severity of squamous metaplasia in ii) was comparable for the test item groups and the vehicle control group. For the changes listed in iii), the number of animals affected was not increased in the test item groups when compared to the vehicle control group. Overall, the above-mentioned changes were observed in all treatment groups including the vehicle control group, and the severity of the changes was similar for the test item groups and the vehicle control group.

Congestion and bronchopneumonia were also observed, but the incidence or severity of the findings did not differ between the treatment groups including the vehicle control group. Of the changes noted in the lungs, vasculitis, calcification, and accumulation of foam cells occurred sporadically, and there were no statistically significant differences in their incidence between the test substance and vehicle control groups.

The following findings were observed in organs other than the lungs: Auricular thrombus in the heart; choledochal cyst, bile-duct hyperplasia, and multiple cysts in the liver; lipidosis in the spleen; keratotic cyst in the skin; lymphocytic hyperplasia in the submucosa of the duodenum and jejunum; whitening of brown fat in the thoracic cavity; and cyst and fat necrosis in the abdominal cavity. However, all these changes were sporadic and there were no statistically significant differences in their incidence between the test item and vehicle control groups.

Relevance of carcinogenic effects / potential:

A mesothelioma was observed in the abdominal cavity of one animal in the high dose group. The mesothelioma originated in abdominal organs and was not accompanied by lung metastasis. In addition, no mesothelial proliferative lesions were noted in any of the other animals in the same group.

The following findings were observed in the test item groups: C-cell adenoma and C-cell adenocarcinoma in the thyroid; neuroblastoma, pheochromocytoma, and malignant pheochromocytoma in the adrenals; islet cell adenoma and islet cell adenocarcinoma in the pancreas; lipoma in the liver; hemangioma and hemangiosarcoma in the spleen; osteosarcoma, nephroblastoma, carcinosarcoma, and malignant fibrous histiocytoma in the abdominal cavity; and malignant lymphoma and myeloid leukemia in the blood vessels of the lungs.

All these findings were sporadic and there were no statistically significant differences in their incidence between the test item and control groups.

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

1.2 other: mg/animal (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 2: Necropsy findings (number of animals with findings)

		Vehicle control (50 animals in total)	Low dose group (49 animals in total)	High dose group (50 animals in total)
Lungs	Black dot	0	1	10
	Black spot	0	1	9
	Blackening	0	2	1
Thoracic cavity	Hydrothorax	16	22	24
	Intrathoracic mass	0	1	0
Abdominal cavity	Ascites	4	7	6
	Intra-abdominal mass	2	0	2
	Intra-abdominal abscess	0	0	0

Table 3: Neoplastic lesions (number of animals with findings)

Organ	Finding	Vehicle control (50 animals in total)	Low dose group (49 animals in total)	High dose group (50 animals in total)
Lungs	Adenoma	0	0	0
Thyroid	C-cell adenoma	0	0	0
	C-cell adenocarcinoma	0	0	0
Adrenals	Neuroblastoma	1	0	0
	Pheochromocytoma	0	0	1
	Malignant pheochromocytoma	0	0	1
Pancreas	Islet cell adenoma	1	0	1
	Islet cell adenocarcinoma	1	0	0
Liver	Lipoma	1	0	0
Spleen	Hemangioma	1	2	3
	Hemangiosarcoma	0	1	1
Intra-abdominal mass	Osteosarcoma	0	0	0
	Nephroblastoma	0	0	0
	Carcinosarcoma	1	0	0
	Malignant fibrous histiocytoma	0	0	0
	Mesothelioma	0	0	1
	Malignant lymphoma	0	1	1*
	Myeloid leukemia	2	0	0

*: In the pulmonary vessel lumen

 

Applicant's summary and conclusion

Conclusions:

Findings such as emaciation were noted in animals that died during the study period. No statistically significant decrease in body weight was found in any of the test item groups compared with the control group. Necropsy of treated animals showed blackening of the lungs, hydrothorax, intrathoracic masses, ascites, and intra-abdominal masses and abscess.

All neoplastic lesions noted in the test item groups were sporadic and no statistically significant difference in their incidence was found between the test substance and control groups. Therefore, all neoplastic lesions observed were considered to be the spontaneous, background lesions of the test animals used and thus were not considered to be induced by the test substances.

In conclusion, the test item was not carcinogenic when administered by intratracheal instillation under the conditions of this study.