Cobalt(2+) disulphamate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt(2+) disulphamate

EC Number:

237-834-1

EC Name:

Cobalt(2+) disulphamate

Cas Number:

14017-41-5

Molecular formula:

Co(H2NO3S)2

IUPAC Name:

cobalt(2+) disulphamate

Test material form:

solid

Details on test material:

- State of aggregation: red-brown solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +15 to 25°C, store in the tighly closed original container, in a cool, dry and well-ventilated place.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: corneae were isolated and used on the same day after delivery of the eyes

Test system

Vehicle:

other: 0.9 % NaCl in deionised water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: 20 % solution (w/v) in vehicle (using sonication for 10 minutes)

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not required

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

PREPARATION OF CORNEAS
- each isolated cornea was mounted according to the description given in OEDC guideline 437, i.e. in a specially designed cornea holder that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium cMEM (MEM, supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin and 1 % fetal calf serum).
- for equilibration, the corneae in the holder were incubated in a vertical position for one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
- all eyes were carefully examined macroscopically for defects before removing the cornea. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- at the end of the equilibration period of the corneae in the holder, the basal opacity was determined (t0).
- corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period were not used.

APPLICATION DOSE AND EXPOSURE TIME
- the anterior compartment received the test item solution or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae.
- corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 240 minutes).
- after exposure of the test item or control items to the corneae, they were rinsed off from the application sides with EMEM containing phenol red at least three times or more if phenol red is still discoloured (yellow or purple), or the test item is still visible.
- once the medium was free of the test item the corneae were given a final rinse with cMEM without phenol red.
- fresh cMEM was added into the anterior compartment and opacity was measured (t240).
- permeability of the corneae was determined.
- no tissue peeling, residual test chemical and non-uniform opacity patterns were observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the opacitometer (OP_KiT opacitometer (Electro Design)) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Evaluation of opacity:
- the change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
- the average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (Versamax® Molecular Devices)(OD490).
- after the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 0.5% (w/v) sodium fluorescein solution in HBSS.
- corneae were incubated in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment was removed, mixed and the optical density at 490 nm was determined with a microplate reader.
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value of permeability determination)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – mean opacity of the negative control) + (15 x (permeability value - mean permeability of the negative control))
The mean IVIS value of each treated group is calculated from the respective individual IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following category according to OECD guideline 437 (please refer to table 1 in the field "Any other information on material and methods incl. tables" below).

DECISION CRITERIA:
The test will be acceptable if:
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- a single testing run composed of at least three corneas should be sufficient for a test chemical when the resulting classification is unequivocal. In cases of borderline results in the first testing run, a second testing run will be considered as well as a third one in case of discordant mean IVIS results between the first two testing runs.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Value:

0.56

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- relative to the negative control, the test item cobalt(II) sulfamate tetrahydrate did not cause an increase of the corneal opacity or permeability.
- after exposure to the negative control (physiological saline) an increase of opacity or permeability of the corneae was not observed (mean IVIS = 0.83).
- exposure to the positive control (10% (w/v) Benzalkonium chloride in saline) resulted in clear opacity and distinctive permeability of the corneae (mean IVIS =120.38) corresponding to the classification as serious eye damaging (EU CLP/UN GHS Category 1).
- the test is valid since the IVIS of the positive control falls within two standard deviations of the current historical mean. Further, opacity and permeability of the negative control are less than the respective established upper limits for background opacity and permeability.

Please refer to the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Table 1: Results after 240 Minutes Treatment Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	0	0.00	0.060	0.055	0.90	0.83	No Category
	0		0.051		0.77
	0		0.054		0.81
Positive Control	121.00*		0.041**		121.62	120.38	Category 1
	132.00*		0.045**		132.68
	104.00*		0.190**		106.85
Cobalt(II) sulfamate tetrahydrate	1.00*		-0.007**		0.90	0.56	No Category
	1.00*		-0.007**		0.90
	0.00*		-0.007**		-0.11

* final corrected opacity ((t240 - t0) - average-corrected negative control opacity value)

** final corrected OD490 (OD490 - average-corrected negative control OD490 value)

Table 2: Historical Data

	Positive Control	Negative Control
Mean IVIS	117.58	1.31
Standard Deviation of IVIS	9.25	0.20
Range of IVIS	98.30—138.03	0.86—1.64
95 % Control limits of IVISpos	99.09—136.08
Mean Opacity t240min	116.18*	0.23
Standard Deviation of Opacity t240min	14.70*	0.20
Range of Opacity t240min	82.00—187.00*	0.00—0.67
Mean Permeability OD490	0.09**	0.07
Standard Deviation of Permeability OD490	0.11**	0.01
Range of Permeability OD490	-0.01—0.52**	0.06—0.09
Values of 59 studies with solid test items sharing 32 sets of controls, performed between January 2016 and February 2018.

* corrected opacity value

** corrected permeability value

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current valid OECD 437 study and under the experimental conditions reported, cobalt(II) sulfamate tetrahydrate should not be categorized (EU CLP/UN GHS No Category). The test item is not eye irritating and not serious eye damaging.