Copper, diamminebis(nitrato-kO)

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

yes

Remarks:

the deviation is related to the acceptability criteria and has an impact of the conclusion of the study (see below)

Qualifier:

according to guideline

Guideline:

other: method B.40bis of the Council regulation No. 440/2008

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

epiCS

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

The reconstituted epidermis measured 0.6 cm².
The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The insert was placed in a 6 wells culture plate which had been previously filled with 1 mL of culture medium. The culture dishes were incubated at 37±2°C, 5% CO2 during 20 hours and 20 minutes before treatment. Just before the treatment, the culture medium was replaced by a new culture medium.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied as supplied, at the dose of 25 mg.

Duration of treatment / exposure:

during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C

Number of replicates:

2 living human skin models

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean percent viability of the epidermis skin treated during 3 minutes at room temperature

Value:

ca. 127.31

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean percent viability of the epidermis skin treated during 1 hour at 37°C

Value:

ca. 59.29

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The item is to be classified as non-corrosive: if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15 %
As the difference of viability between the two treated tissue replicates for the 1 hour treatment, was >30% (87.4%), the acceptability criteria define in OECD 431 are not respected. Moreover one epidermis treated with the test item presented with a viability at 15.60%, which is very closed to the cut off for the classification as corrosive. A second run is necessary to conclude on the corrosive effect of the test item.

Interpretation of results:

study cannot be used for classification

Conclusions:

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 127.31 % and 59.29% (102.99% in one epidermis and 15.60% in the other one), versus 8.68% and 0.17%, respectively, with the positive control item (potassium hydroxide 8N).

As the difference of viability between the two treated tissue replicates for the 1 hour treatment, was >30% (87.4%), the acceptability criteria define in OECD 431 are not respected. Moreover one epidermis treated with the test item presented with a viability at 15.60%, which is very closed to the cut off for the classification as corrosive. A second run is necessary to conclude on the corrosive effect of the test item.

Reference 2

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)

Deviations:

yes

Remarks:

without impact on the conclusion of the study.

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

Episkin

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

The reconstructed epidermises (Episkin) measured 0.50 cm². The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium during 2 hours and 30 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 μL of maintenance medium.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied as supplied, at the dose of 16 mg.

Duration of treatment / exposure:

during 42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

41 hours and 35 minutes post-treatment incubation period

Number of replicates:

3 living human skin models

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

ca. 87.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Validity criteria were fulfilled for the negative and the positive controls.
The difference of viability between the 3 tissues treated with the test item was 24.6%, instead of 18% at the maximum as initially scheduled. Considering the results obtained (each epidermis is clearly classified as “Non-irritant to skin” with viability > 50%), this deviation is considered as without impact on the conclusion of the study.

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item MNP-363H has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.
No hazard statement or signal word is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye 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 438 (Isolated Chicken Eye 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

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Details on test animals or tissues and environmental conditions:

The eyes collected from chickens obtained from a slaughterhouse where they are killed for human consumption have been used for this assay.
The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were thereafter enucleated.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg of the test item was applied

Duration of treatment / exposure:

during 10 seconds

Observation period (in vivo):

After the application of the test item, the eyes were rinsed with 40 mL of physiological saline. The product still stuck to the entire cornea in the three eyes and was finally removed using a physiological saline soaked cotton bud then rinsed again with 10 mL of physiological saline.

Number of animals or in vitro replicates:

3 enucleated chicken eyes

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

ca. 2.3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

ca. 2.3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

mean

Value:

ca. 3

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.
The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 2.3, corresponding to ICE class III;

- mean score of fluorescein retention: 2.3, corresponding to ICE class III;

- maximal mean corneal swelling: 3%, corresponding to ICE class I.

The combination of the three endpoints for the test item MNP-363H was 2 x III, 1 x I.

Interpretation of results:

study cannot be used for classification

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item MNP-363H is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.

Reference 2

Endpoint:

eye 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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

yes

Remarks:

without impact on the conclusion of the study

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

The Reconstructed human Cornea-like Epithelia (EpiOcular™) measured 0.60 cm².
The same day, the tissues in their well shipping container were equilibrated to room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 20 hours and 27 minutes at standard culture conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied as supplied at the dose of 50 mg.

Duration of treatment / exposure:

During 6 hours

Duration of post- treatment incubation (in vitro):

18 hours post-exposure incubation

Number of animals or in vitro replicates:

2 living RhCE tissue replicates

Details on study design:

After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DP BS. The rinsed tissues were checked for any coloration and noted to be of comparable colour with the negative control treated tissues (whitish). This rinsing step was followed by a 25-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue.
The RhCE constructs were then incubated for an 18 hours post-exposure incubation at standard culture conditions in 1 mL offresh medium at 37°C, 5% CO2.

Irritation parameter:

other: % tissue viability

Run / experiment:

mean

Value:

ca. 2.76

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Deviation to the study plan:
Acceptability criteria: The difference of viability between the 2 tissues treated with the negative control was 50.16%, instead of 20% at the maximum as initially scheduled. This was due to an aberrant value obtained with one negative control epidermis (0.369), which was under the historical range of values. This value was not taken into account in the calculation of cell viabilities. Considering the results obtained, this deviation is considered as without impact on the conclusion of the study (the fact to take into account or not this value does not change the classification of the test item).

Interpretation of results:

other: The test item is classified but the study cannot distinguish between Category 1 or Category 2

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item MNP-363H has to be identified as potentially requiring classification and labelling according to UN GHS Category 2 or Category 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test substance is not classified for skin irritation, based on the negative results observed in the in vitro skin irritation study.

Regarding eye irritation, the test substance is positive in the in vitro eye irritation study so it has to be classified either in Category 1 or in Category 2 for the eyes.

Based on the fact that the in vitro eye corrosion study cannot conclude on the corrosive potential of the substance and that the skin irritation study is negative, the substance is classified Eye Irrit 2 (Category 2).