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Administrative data

Description of key information

Skin corrosion: not corrosive (OECD 435; GLP)

Skin irritation: irritating to skin (OECD 439; GLP)

Eye irritation (OECD 437): not serious eye damaging (CLP (Cat 1) and not serious eye irritating. IVIS score < 3

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MatTek Corporation Protocol: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)
Version / remarks:
2014-11-07
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14
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 tightly closed original container, in a cool, dry and well-venitlated place
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: humans
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
Justification for test system used:
In a prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
other: Dulbecco's phosphate buffered saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the tissues were rinsed with PBS at least 15 times in order to remove any residual test material.

After the rinsing the inserts were submerged in PBS at least three times. Afterwards the inserts were again rinsed with PBS. The tissues were then transferred into plates with assay medium. Tissues were incubated for 24 ± 2 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation medium was changed (pre-warmed fresh medium). Thereafter tissues were incubated for another approx. 18 ± 2 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was maximal 42 ± 4 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after the incubation period, the tissues were rinsed three times with PBS. The tissues were transferred into new plates containing extractant solution (isopropanol) in each well ensuring that the tissues were completely covered and the plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for at least 2 hours while shaking at room temperature or overnight at room temperature without shaking or up to 72 hours without shaking in the refirgerator.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken and the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate. The optical density was determined with a plate spectrophotometer. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm

TEST FOR COLOUR INTERFERENCE
Before the test started, functional check for colour interference was performed. 25 ± 2 mg of the test item were added to deionised water. The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 minutes. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If solution changes colour significantly, the test item is persumed to have the potential to stain the tissue. A functional check on viable tissues should be performed.
To check the tissue-binding of a coloured test item (or a test item that changes into a coloured test item), two viable tissues were exposed to 25 ± 2 mg of the test item. In parallel, two tissues were exposed to DPBS (negative control). All procedures were followed as described above for the MTT assay except the tissue was incubated for 3 hour incubation in culture media without MTT at standard incubation conditions instead of incubating in media containing MTT. After the 3 hour incubation, the tissues were rinsed and the tissues were extracted using isopropanol and the optical density (OD) at 570 nm was measured.
Data correction procedure:
If the tissues treated by coloured test item (or test item detected in step 1) have a mean optical density between 5% and 30% of the negative control tissue (treated with DPBS), the real MTT optical density (unaffected by interference with the coloured test items) is calculated using following formula:
OD = ODcoloured tissue (MTT assay) – ODcoloured tissue (no MTT assay)
If the tissues treated by coloured test item (or test item detected in step 1) has an OD < 5% of the DPBS treated control tissue and the tissue viability (determined in MTT assay) is not close to the classification cut-off (50%), correction of the results is not necessary.
If OD of from the tissue treated by coloured test item (or test item detected in step 1) is > 30% of the DPBS treated control tissue, additional steps and expert judgment must be performed to determine if the test item must be considered as incompatible with the test.

TEST FOR DIRECT MTT REDUCTION
The test item was evaluated for its potential to interfere with MTT assay. To test if a test item directly reduces MTT, 25 ± 2 mg of the test item were added to 1 mL of the MTT-solution (1 mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: relative viability(%) = (mean OD test item or positive control/ mean OD of negative control) x 100
For the test item and the positive control, the mean relative viability ± standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritation potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 25 ± 2 mg (~ 39 mg/cm²) of the test item, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5 % Sodium dodecyl Sulfate (SDS) solution
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
maximal 42 ± 4 hours
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Value:
20.9
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: the test item did change colour in the presence of water. A red colouration was observed. An additional test with viable tissues (but without MTT addition) was necessary to be performed.
- Direct-MTT reduction: the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test item is irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is irritating to the skin (Category 2: H315).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
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
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
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.
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
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.
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.

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

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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin corrosion/irritation:

The substance does possess a skin irritation potential based on an in vitro OECD 435 and 439, respectively. Cobalt(2 +) disulfamate does require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations (Category 2; H315).

Eye irritation:

According to a OECD 437 guideline study (GLP), cobalt(2 +) disulfamate is neither serious eye damaging (CLP (Cat 1) nor serious eye irritating. Hence, it does not require C&L as eye irritating in accordance with Regulation (EC) No 1272/2008 and its subsequent adaptations.