Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

EC Number:

611-056-6

Cas Number:

538313-26-7

Molecular formula:

C2H6CuN10

IUPAC Name:

Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Provider : AUTOLIV ASP, 16700 W. Hwy 83, Promontory, UTAH 84307, USA
- batch No. P3656461
- Expiration date of the lot/batch: august 25th 2020
- Purity: 97.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a restricted area dedicated to explosive products, at outside temperature and humidity
- Solubility and stability of the test substance in the solvent/vehicle: not soluble in water or other tested vehicle, homogeneous suspensions

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no treatment

FORM AS APPLIED IN THE TEST (if different from that of starting material)
homogeneous suspensions in vehicle

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: bovine eye were obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, FRANCE
- Age : bovine cattle were up to 12 months old

Test system

Vehicle:

other: parafin oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750µL of test item 20% (w/v) in vehicle (parafin oil)
homogeneous blue suspension

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

0

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): On completion of the treatment period, the test item formulation was removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed as follows:
- any test item formulation adhering to the walls of the anterior chamber was removed with a cotton bud and using a pipette of heated cMEM (32°C),
- the corneas were rinsed three times with pre-warmed cMEM containing phenol red (i.e. until the test item formulation had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.
The rinsing efficiency was visually confirmed by observing the transparency and the colour changing of the rinsing medium (containing phenol red). Some difficulties were encountered during the rinsing. Indeed, residual amounts of test item formulation were noted on the walls of the anterior chamber but were finally removed.

SCORING SYSTEM:
The following formula was used to determine the In Vitro Irritancy Score (IVIS):
IVIS = (OPT2-OPT0) + (15 x cOD490 nm)
The IVIS was calculated for each test item formulation and positive control cornea. The mean IVIS for each series of three corneas was calculated from the individual scores.


TOOL USED TO ASSESS SCORE:
OPACITY MEASUREMENTS: An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded. Just before the first opacity measurement (i.e. OPT0), the opacitometer was calibrated using specific calibrators. Values obtained for each calibrator were as follows:
- calibrator No. 1: set to 75,
- calibrator No. 2: from 145 to 155,
- calibrator No. 3: from 218 to 232.
Just before the second opacity measurement (i.e. OPT2), the opacitometer was calibrated using the calibrator No. 1 set to 75. Just after each opacity measurement (OPT0 and OPT2), the calibration of the opacitometer was checked by using the calibrator No. 1. The obtained value was between 73 and 77.
Corneal opacity was determined by reading each holder in the right hand chamber of the calibrated opacitometer, versus an empty holder (without cMEM, cornea and glasses), placed in the left hand chamber.
For opacity measurement, care was taken to make sure that no air bubbles were present within the holders containing corneas (by ensuring that each compartment was filled to overflowing with heated cMEM) and each holder was wiped dry.

PERMEABILITY DETERMINATION
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a 5 mg/mL fluorescein solution in cMEM.
The Optical Density at a wavelength of 490 nm (OD490 nm) of this dilution was measured. As the value obtained was between 0.850 and 0.940 the fluorescein solution was validated.
For each series of three corneas, a chronometer started from the fluorescein solution application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes).
At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).

MACROSCOPIC EXAMINATION
After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium (see Table 1). Then, the corneas were discarded.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control

Value:

173

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: in the range 2 SD of historical data

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

test item 20%

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean opacity <4.4 and mean fluorescein OD490nm <0.0296
- Acceptance criteria met for positive control: the mean IVIS should fall within 2 SD of the historical mean (105.2 to 186)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, CuDABT, was identified as a test item not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, CuDABT, to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. The design of this study was based on the guideline OECD Guideline 437 and the study was performedin compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Method

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes at +32°C.

A single experiment was performed using three corneas for each treated series (test item formulation, positive and vehicle controls).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item was applied at the concentration of 20% (w/v) in the vehicle (paraffin oil), in a single experiment using a treatment time of 4 hours and using the open-chamber method. Vehicle and positive controls were applied using the same treatment time and using the closed-chamber method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. A second opacity measurement was then performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes at +32°C. At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

Results

Macroscopic examinations

No notable opaque spots or irregularities were observed on all test item formulation-treated corneas.

In Vitro Irritancy Score

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The mean In Vitro Irritancy Score (IVIS) of the test item formulation-treated corneas was: 1.

As the mean IVIS was < 3, the test item was considered asa test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Conclusion

Under the experimental conditions of this study, the test item, CuDABT, was identified as a test item not requiring classification for eye irritation or serious eye damage (UN GHS No Category).