3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-10 to 2019-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, no. 437 (adopted: 09 October 2017)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Munich, Germany

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane

EC Number:

202-928-3

EC Name:

3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane

Cas Number:

101-25-7

Molecular formula:

C5H10N6O2

IUPAC Name:

3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane

Test material form:

solid

Details on test material:

- Name: 3,7-Dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane
- CAS: 101-25-7
- Batch: 458231 GDSK
- MW: 186.17 g/mol
- Purity: 95.5 %
- Appearance: beige solid
- Stability: instable after repeated contact to light; undergoes hydrolysis at acidic pH
- Expiry Date: 31 July 2019
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals / tissue source

Species:

other: isolated bovine corneas

Details on test animals or tissues and environmental conditions:

The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

The test item was ground to a powder in a mortar with pestle and subsequently suspended with physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 1801168, expiry date: 12/2020) to give a 20% concentration.

Duration of treatment / exposure:

After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red).

Duration of post- treatment incubation (in vitro):

90 min fluorescein incubation

Number of animals or in vitro replicates:

3 per test group

Details on study design:

CALIBRATION OF THE OPACITOMETER
The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 95-105 lux. The calibration procedure was performed before the test and is documented in the raw data.

TREATMENT OF THE CORNEAS
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI 1640 medium. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.
750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI 1640 medium. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

4 h treatment

Value:

0.52

Vehicle controls validity:

valid

Remarks:

0.34

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

84.28

Results 2

Irritation parameter:

cornea opacity score

Remarks:

mean corrected value

Run / experiment:

4 h treatment

Value:

0.53

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

57.81

Results 3

Irritation parameter:

other: Permeability

Remarks:

mean OD490

Run / experiment:

4 h treatment

Value:

0.011

Vehicle controls validity:

valid

Remarks:

0.012

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

1.777

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to the evaluation criteria the test item is classified into UN GHS "No Category".

Executive summary:

Summary Results

The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item: The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration.

Visual Observation after treatment:  None of the corneas showed any opacity of the tissue.

Mean in vitro irritation score: 0.52

X	UN GHS No Category
	No prediction can be made
	UN GHS Category 1

Classification

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Conclusion

According to the evaluation criteria the test item is classified into UN GHS "No Category".