産総研：安全科学研究部門サイト > Development of Innovative Methodology for Safety Assessment of Industrial Nanomaterials > Research Activities and Results (June 2015) > Research and development item: ②(b-1) Study on standardization of intratracheal administration testing: Study on standardization of the skill

Research and development item: ②(b-1) Study on standardization of intratracheal administration testing: Study on standardization of the skill

Implemented by the Chemicals Evaluation and Research Institute

Final objective: We compile and release a draft standard procedure manual for intratracheal administration testing, including a method for verifying the skills of intratracheal administration testing engineers.

Main results:
Establishment of the standard intratracheal administration testing procedure

To establish the standard intratracheal administration testing procedure, we conducted intratracheal administration testing of nanomaterials under various testing conditions to study the effect of differences in testing conditions on the test results centered on pulmonary toxicity (inflammatory reaction). In this research and development item, we used BALF results as an endpoint to quantitatively compare the degree of pulmonary toxicity.

Prior to the study, to minimize fluctuation among BALF samples collected by different operators, we standardized a BALF collection method. First, to lavage the inside of the alveoli, we inserted and ligated a catheter into the trachea, and then injected 7 mL of lavage fluid (sterilized saline) through the catheter. At this time, to maintain a constant injection pressure, we fixed the height of the saline level at the start of injection to 30 cm above the catheter and then allowed the saline drops to fall free (the injection pressure of the lavage fluid was 30 cm H2O). We recovered the injected lavage fluid by free fall from the catheter. We repeated the lavage and recovery twice. This BALF collection method enabled us to minimize fluctuation among lavage samples and steadily recover approximately 90% of the injected lavage fluid irrespective of the operator.

In this study, we used the aforementioned BALF collection method and changed the administration instruments, quantity of the administered fluid, and anesthesia at dissection to conduct intratracheal administration testing and establish a range of testing conditions and an anesthesia method that do not affect the BALF testing results.

Method of verifying skills of intratracheal administration testing engineers
Intratracheal administration operation includes several noteworthy steps such as insertion of an administration instrument into the trachea and control of the insertion depth; poor skill or technique on the part of the administrator may result in improper assessment of pulmonary toxicity. Therefore, a method of verifying the administrator’s skill is needed. Focusing on incorrect administration factors in intratracheal administration operation, we developed a method (draft) of verifying the skills of intratracheal administration testing engineers (Table ②(b-1)). The skill verification method consists of an initial stage that does not require dissection of animals taking animal protection into consideration; this stage is intended to improve the level of skill associated with the insertion operation of an administration instrument into the trachea. In the second stage, dye solution and nanomaterials of known toxicity are administered into the trachea, and the samples are dissected to determine the result of administration visually or using inflammatory reaction as an endpoint.

Table②(b-1) Method of verifying skills of intratracheal administration testing engineers

Skill item Skill verification point
1st stage
(dissection unnecessary)		 Improve the level of skill associated with laryngoscope operation.
  • Understand the anatomical structure of the larynx and use a laryngoscope to visualize an instrument insertion point (epiglottis).
Perform sequential operations up to smoothly inserting the instrument.
  • Before the animal comes out from the anesthetic, retention, a visual verification of the epiglottis, and instrument insertion are completed.
Correctly insert an administration instrument into the trachea.
  • Insert an instrument without disturbing the surrounding mucous membranes at insertion.
  • Confirm that the administration instrument is inserted into the trachea by touching the tracheal cartilages with the instrument.
Insert an administration instrument into an appropriate position and perform administration.
  • Confirm that the administration instrument is inserted into the appropriate position.
  • Administer medium and listen to moist rale for a given length of time immediately after administration.
  • Check that blood does not attach to the administration instrument after administration.
2nd stage
(dissection necessary)		 Administer fluid so that the fluid is not localized in the lung.
  • Administer a dye solution and visually verify that backflow does not occur immediately after administration.
  • Dissect the animal after administration of the dye solution to check distribution to the right and left lungs.
Administer medium into the trachea without inducing a biological response change.
  • By BALF testing or another procedure, confirm that the results are similar to existing research information or testing results conducted under the same conditions.
Confirm reproducibility of results in intratracheal administration testing.
  • Administer materials of known toxicity and confirm that results similar to existing research information are obtained.