Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

EC Number:

284-943-5

EC Name:

Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

Cas Number:

84989-53-7

Molecular formula:

C42H44CoN12.Cl

IUPAC Name:

hydroxylamine

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Test system: Freshly isolated bovine cornea
- Source: abattoir A. Moksel AG, Buchloe, GermanyA. Moksel AG, Buchloe, Germany

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

Anterior compartment received the test item or vehicle or positive contril at volume of 0.75 mL on the surface of the cornea.

TEST MATERIAL
- Amount(s) applied (volume): 0.75 mL
- Concentration (if solution): test item suspended with physiological saline 0.9% NaCl to give a 20% concentration.

Duration of treatment / exposure:

4 hours ± 5 minutes incubation at 32 ± 1 °C.

Duration of post- treatment incubation (in vitro):

After illuminance measurement was performed incubated for 90 minutes at 32 ± 1 °C.

Number of animals or in vitro replicates:

3 corneas per dose group

Details on study design:

Preparation of the Corneas
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.

Treatment of the Corneas
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. 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.

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

Evaluation of Results
The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer: Opacity = ((Io/I)-b)/a
with a = 0.025 and b = 0.9894

The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

>= 122.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: classified into UN GHS Category 1.

Other effects / acceptance of results:

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 should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

In vivo

Irritant / corrosive response data:

All 3 corneas treated with Ingrain Blue 5 showed a blue coloration of the tissue.
The following mean in vitro irritation score was calculated: 122.10

Any other information on results incl. tables

In vitro irritation score

Cornea No.	Test Item	Corrected Opacity	Permeability Corrected OD490 Value	IVIS
1	Negative Control	0.89	0.009	0.59
2		0.04	0.009
3		0.04	0.036
MV		0.32	0.018
4	Positive Control	90.91	1.417	121.72
5		92.93	2.397
6		89.52	2.307
MV		91.12	2.04
7	Test Item	161.93	-0.013	122.1
8		125.17	-0.014
9		79.84	-0.015
MV		122.31	-0.014

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The mean in vitro irritation score was 122.1 %. It is hence concluded that the test item can lead to irreversible eye damage.

Executive summary:

The eye irritancy potential of Ingrain Blue 5 was investigated in the bovine corneal opacity and permeability assay according to OECD guideline 437 and in compliance to GLP. The test item was suspended with physiological saline 0.9% NaCl (see 10.2) to give a 20% concentration and 3 corneas were treated.

All 3 treated corneas treated showed a blue coloration of the tissue. The following mean in vitro irritation score was calculated: 122.10. Based on these results, the test item was classified as eye damage 1 according to CLP.

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.