Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-08-01 to 2010-05-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted according to provisions laid down in GLP; however since it was intended a pre-experiment giving information on intensity and reversibility of effects no inspections by the Quality Assurance Unit have been done. No OECD guideline available. Study is well documented and fully reliable.

Data source

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
- Two different concentrations of the test item were intratracheally instilled
- Effects and Recovery were compared either to Quartz or saline (vehicle)
- Body weights, wet lung weight, as well as BAL parameters were chosen as endpoint
- Gross pathology/necropsy was also performed
GLP compliance:
yes
Remarks:
Study followed the principles of GLP; however, since this study was intended to be a pre-experiment no inspections by the Qualitiy Assurance Officers have been done. GLP-certificate is attached to full study report. Study was fully compliant with the Regu

Test material

Radiolabelling:
no

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Age at study initiation: 5-6 weeks of age
- Weight at study initiation: 179-180g
- Housing:
- Individual metabolism cages: yes/no
- Diet (e.g. ad libitum): ad libitum (ssniff V1534, ssniff Spezialdiäten Soest, Germany)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Photoperiod (hrs dark / hrs light): 12hrs/12hrs

Administration / exposure

Route of administration:
intratracheal
Vehicle:
physiological saline
Details on exposure:
- Rats were shortly anesthesized by carbon dioxide
- Test item suspended in 0.3mL physiological saline was administered using a cannulua
- The total dose was given on two consecutive days in 2 aliquots (half of the total dose each) to assure the homogeneous distribution of the test material in the lungs
Duration and frequency of treatment / exposure:
- 3 and 14 days after the administration of the test items the animals were killed for BAL analysis
Doses / concentrations
Remarks:
Doses / Concentrations:
0.5mg per rat and 3mg per rat (suspended in 0.3mL physiological saline)
No. of animals per sex per dose:
5 rats per dose per group
Control animals:
yes, concurrent vehicle
Positive control:
Quartz (DQ12) served as positive control; concentration = 0.3mg per rat (5 rats per dose per group)
Details on dosing and sampling:
- Bronchoalveolar lavage was performed in 5 female rats per time point and group, i.e. 3 days after the last instillation and after 14 days of post-observation.
- Lungs were lavaged with saline using two lavages of 4mL each
- Lavage fluid was kept on ice
- Leukocyte concentration of the lavagate was determined using a counting chamber and two cytoslides were prepared with a cytocentrifuge for differential cell count
- After centrifugation of the lavage fluid, biochemicalindicators relevant for diagnosisof lung damage were determined in the supernatant (LDH, b-glucoronidase, total protein)
- These parameters were analysed according to routine clinical chemistry protocols using a Cobas Fara device
- Please also refer to section "Any other information on materials and methods incl. tables"
Statistics:
ANOVA and Dunnet's test

Results and discussion

Any other information on results incl. tables

Survival and Clinical Observations

- After treatment, the rats were carefully inspected. During the anesthesia/instillation procedure single animals had to be ventilated directly after administration of test/reference items.

- Thereafter, the general health status of the animals was within normal limits.

- No deaths occurred during the anesthesia and intratracheal instillation procedure.

- No deaths were observed in the observation period till sacrifice in any of the groups (vehicle control or particle-treated).

- The macroscopical observations made upon sacrifice of animals reflect some test item-related effects such as the particle deposition in the deep lung areas (discoloration), however, severe test item-related adverse effects were not observed macroscopically

Body weight

- Statistically significant differences of body weight, as compared to vehicle controls, were observed only in the Expanded Graphite high dose group on day 3 after the intratracheal instillations.

- This decrease is considered as a test item-related effect, however, the body weight development has returned to normal already on day 7 of the recovery period.

Terminal Body and Lung Wet Weights - (BAL rats)

- Absolute and relative wet lung weights were statistically significantly elevated in the Graphite groups after 3 days as compared to the vehicle control.

- After 14 days the absolute and relative lung weights of the Expanded Graphite high dose group were still statistically significantly different from the vehicle control group.

Bronchoalveolar Lavage (BAL)

- The vehicle control shows values that were at historical baseline levels.

- LDH, β-glucuronidase and total protein showed dose-dependent increases in the Graphite Powder groups on day 3 with clear recovery effects on das 14.

- The Positive Control Quartz DQ12 induced a more pronounced response than the graphite low dose groups.

Total and Differential Cell Counts for Expanded Graphite:

- PMN levels of 12.2% (low dose) and 27.3% (high dose) on day 3 after treatment.

- After 14 days of recovery for these values a decrease to 10.9% and 22.8%

- Expanded Graphite showed a slight inflammatory effect in the low dose group and a moderate effect in the high dose group.

- After 14 days a slight recovery was observed, however, the PMN levels were still statistically significantly increased.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: At the low dose Graphite showed slight inflammatory effects that recovered partially within 14 days. Based on these findings, the recovery period for the repeated dose inhalation studies should be prolonged to 28d.
Executive summary:

- Two different concentrations of the test item were intratracheally instilled

- Effects and Recovery were compared either to Quartz or saline (vehicle)

- Body weights, wet lung weight, as well as BAL parameters were chosen as endpoint

- Gross pathology/necropsy was also performed

- All animals survived the study

- Effects indicating systemic toxicity were not observed

- At the low dose Graphite showed slight inflammatory effects that recovered partially within 14 days