Copper Chelate Complex

Endpoint summary

Description of key information

Skin corrosion (in-vitro):

An in vitro skin corrosivity test was conducted on the test item in a reconstructed human epidermis model according to OECD test guideline 431 and EU-method B40. Following exposure to the test item the mean cell viability after 3 minutes of treatment was 100.8 %, the mean cell viability after 1 hour of treatment was 86.8% compared to the negative control. These are above the threshold values in both cases, therefore the test item was considered to be non-corrosive to skin under the conditions of this assay. The experiment met the validity criteria, and therefore the study was considered to be valid. In conclusion, under the conditions of this in vitro EpiDerm™ SCT Model corrosivity assay, the results indicate that the test item is non-corrosive.

 

Skin irritation (in-vitro):

An in vitro skin irritation test was conducted on the test item in a reconstructed human epidermis model according to OECD test guideline 439 and EU-method. B 46.Following exposure of the EpiDerm Model to the test item, the mean relative viability was 3.8% compared to the negative control value. This is below the threshold of 50%, therefore under the condition of this assay the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EpiDerm model test with the test item, the results indicate that the test item is irritant to skin, GHS Classification: Category 2 (since the test item is known to be non-corrosive).

 

Eye irritation (in-vitro):

An in-vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline. The test item showed no corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Minimal amount of test item (negligible) was observed on the corneal surfaces any eyes in experiments at 240 minutes after the post-treatment rinse. This fact had no impact classification of the test item. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes, the test item was non-irritant, UN GHS Classification: No Category.

Conclusion:

In the in vitro EpiDerm model test with the test item, the results indicate that the test item is irritant to skin, GHS Classification: Category 2 (since the test item is known to be non-corrosive). Based on the available test results, a clear classification was possible, therefore no further testing in-vitro or in-vivo testing was required

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

Study period:

April - June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from light and humidity (stored in a tightly closed container)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: no data

Details on animal used as source of test system:

EpiDerm™ Model (EPI-200-SCT) (Source: MatTek, Bratislava, Slovakia, Batch No.: 30859, Expiry date: 24 April 2020) units consist of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis (0.63 cm2). It’s 3D structure consisting of organized and proliferative basal cells, spinous and granular layers, and cornified epidermal layers are mitotically and metabolically active. Its use for skin corrosivity testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EpiDerm™ Model (EPI-200-SCT) has been validated for corrosivity testing in an international validation study [2] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

water

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- The plates with the treated epidermis units were incubated for the exposure time of 3 minutes and 1 hours at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere.
- The plates with the treated epidermis units were incubated for the exposure time of 25 minutes (± 0.5 minute) at room temperature (23.5-23.8°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the incubation times the EpiDerm™ units were removed and rinsed thoroughly with DPBS solution (= insert was filled and emptied 20 times in a constant soft stream of DPBS in a glass beaker filled with at least 100 mL DPBS solution) to remove all of the test item from the epidermal surface. The rest of the DPBS was removed from the epidermal surface using a pipette (without touching the epidermis).

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1))
- Wavelength: 570 nm
- Spectrophotometer: standard apparatus

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-corrosive to skin if the relative tissue viability after 3-minute treatment with a test item is above 50%.and 1 hour >= 15%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied in this original form; no formulation was required. 25 mg test item was applied to the epidermal surfaces in each exposure time point and 25 μL of distilled water was added for wetting of the test item (to increase tissue surface contact).

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

In this assay, two replicates for the test item were used. Two negative controls and two positive controls were also run. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific colour optical density (OD) evaluation.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

100.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

86.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Following exposure to the 0 test item the mean cell viability after 3 minutes of treatment was 100.8 %, the mean cell viability after 1 hour of treatment was 86.8% compared to the negative control. These are above the threshold values in both cases, therefore the test item was considered to be non-corrosive to skin under the conditions of this assay. The experiment met the validity criteria, and therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was found to be non-corrosive in this in-vitro test.

Executive summary:

An in vitro skin corrosivity test was conducted on the test item in a reconstructed human epidermis model according to OECD test guideline 431 and EU-method. B40. Following exposure to the test item the mean cell viability after 3 minutes of treatment was 100.8 %, the mean cell viability after 1 hour of treatment was 86.8% compared to the negative control. These are above the threshold values in both cases, thereforethe test item was considered to be non-corrosive to skin under the conditions of this assay. The experiment met the validity criteria, and therefore the study was considered to be valid. In conclusion, under the conditions of this in vitro EpiDerm™ SCT Model corrosivity assay, the results indicate that the test item is non-corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mayl - June 2020

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from light and humidity (stored in a tightly closed container)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: no data

Details on animal used as source of test system:

EpiDerm™ Model (EPI-200-SIT) (Source: MatTek, Bratislava, Slovakia, Lot No.:30867, Expiry Date: 22 May 2020) units consist of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis (0.6 cm2). It’s 3D structure consisting of organized and proliferative basal cells, spinous and granular layers, and cornified epidermal layers are mitotically and metabolically active. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EpiDermTM has been validated for irritation testing in an international validation study [3] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

water

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- The plates with the test item, negative and positive control treated epidermis units were incubated for 35 minutes (± 1 minute) to the humidified incubator at 37°C with 5 % CO2, in a >95% humidified atmosphere and for the exposure time of 25 minutes (± 0.5 minute) at room temperature (25.7-26.5°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the incubation time, the EpiDerm TM units were removed and rinsed into clean beakers containing 100 mL of DPBS each (20 times). Any remaining liquid were removed onto an absorbent paper. The rest of the DPBS was removed from the epidermal surface using a pipette (without touching the epidermis

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1))
- Wavelength: 570 nm
- Spectrophotometer: standard apparatus

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item considered to be irritant to skin (Category 2), if the mean relative viability is less or equal (≤) to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

As the test item is solid, first an appropriate amount (25 µL) DPBS was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 25 mg of test item was applied evenly to each of three test units and each additional control skin units. 25 mg of test item was sufficient to cover the epidermal surface.

Duration of treatment / exposure:

25 and 35 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. As the test item was coloured, two additional test item-treated living tissues were used for the non-specific optical density (OD) evaluation.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

3.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Following exposure of the EpiDermTM Model to the test item, the mean relative viability was 3.8% compared to the negative control value. This is below the threshold of 50%, therefore under the condition of this assay the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item was found to be irritant in this in-vitro skin irritation test.

Executive summary:

An in vitro skin irritation test was conducted on the test item in a reconstructed human epidermis model according to OECD test guideline 439 and EU-method. B 46. Following exposure of the EpiDermTMModel to the test item, the mean relative viability was 3.8% compared to the negative control value. This is below the threshold of 50%, therefore under the condition of this assay the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EpiDerm model test with the test item, the results indicate that the test item is irritant to skin,UN GHS Classification: Category 2 (since the test item is known to be non-corrosive).

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:

April - May 2020

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

Principles of method if other than guideline:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from light and humidity (stored in a tightly closed container)

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Source: TARAVIS KFT (Address: 9600 Sárvár, Rábasömjéni utca 129., Hungary)
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Charles River Laboratories Hungary Kft. at ambient temperature at the earliest convenience. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Charles River Laboratories Hungary Kft. and processed within 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg test item

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

Two experiments were conducted. In each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

Details on study design:

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). In each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

Irritation parameter:

percent corneal swelling

Run / experiment:

1

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: up to 75 min; indication of slight irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

1

Value:

-2.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: up to 240 min; ndication of slight irritation

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

2

Value:

-0.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: up to 75 min; indication of slight irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

2

Value:

-1.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: up to 240 min; indication of slight irritation

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

1.33

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: indication of slight irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

2

Value:

0.33

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: indication of slight irritation

Other effects / acceptance of results:

Experiment 1:
Minimal corneal swelling change (mean = -2.7%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (mean = 1.33) was observed. Minimal fluorescein retention change (mean = 0.33) was noted. Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse. No other morphological effect was observed.

Experiment 2:
Minimal corneal swelling change (mean = -1.1%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (mean = 1.33) was observed. Minimal fluorescein retention change (mean = 0.33) was noted. Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse. No other morphological effect was observed.

Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.5%	I
Mean maximum corneal swelling at up to 240 min	-2.7%	I
Mean maximum corneal opacity change	1.33	II
Mean fluorescein retention change	0.33	I
Other Observations	Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	2xI 1xII

  

Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	-0.5 %	I
Mean maximum corneal swelling at up to 240 min	-1.1%	I
Mean maximum corneal opacity change	1.33	II
Mean fluorescein retention change	0.33	I
Other Observations	Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	2xI 1xII

SUMMARY TABLE FOR UN GHS CLASSIFICATION

 

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II:	True
No severe corneal morphological changes:	True
Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse:	False*

 

Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	False
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False

 

Criteria for “No prediction can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study:	True*

*Minimal amount of test item (negligible) was observed on the corneal surfaces any eyes in experiments at 240 minutes after the post-treatment rinse. This fact had no impact classification of the test item.

The test item showed no corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, based on thesein vitroeye irritation tests in isolated chicken eyes, the test item was non-irritant, UN GHS Classification: No Category.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these in vitroeye irritation tests in isolated chicken eyes, the test item was found to be non-irritant (UN GHS Classification: No Category).

Executive summary:

An in-vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline. The test item showed no corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Minimal amount of test item (negligible) was observed on the corneal surfaces any eyes in experiments at 240 minutes after the post-treatment rinse. This fact had no impact classification of the test item. Therefore, based on thesein vitroeye irritation tests in isolated chicken eyes, the test item was non-irritant, UN GHS Classification: No Category.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In the in vitro skin irritation test acc. OECD 439 (EpiDerm model test), the test item was found to be irritant to skin, GHS Classification: Category 2 (since the test item is known to be non-corrosive).