copper sulfate;N'-[2-(2-aminoethylamino)ethyl]ethane-1,2-diamine

Endpoint summary

Description of key information

Information on skin irritation/corrosion and eye irritation was available for two category members. Based on this information, the substance was classified as Skin irritant (Cat. 2) and for irreversible damage to the eyes (Cat. 1).

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:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
See the documentation for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation for the category approach.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS, Cell Systems
- Tissue batch number(s): Batch No.100-AG2206-1)
- Production date: 27 November 2017
- Shipping date: 27 November 2017
- Delivery date: 28 November 2017
- Date of initiation of testing: 28 November 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2,
- Temperature of post-treatment incubation (if applicable): for 2 hours 55 minutes between 36.9°C and 37.8°C, 5% CO2..

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 3 minutes and 1 hour after the test item application, the human epidermis was washed 20 times with 1 mL of DPBS (DPBS – Dutscher, Batch No. 2780917).
- Observable damage in the tissue due to washing:
The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues.
- Modifications to validated SOP: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time:
The skin samples were placed in 300 μL of a MTT solution at 1 mg/mL for 2 hours and 55 minutes between 36.3°C and 37.4°C, 5% CO2.
- Spectrophotometer: not specified
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function: 55600%
- Morphology: sufficient cornified (5) and vital (4) cell layers
- Contamination: no indication of HIV1, HBV, HCV, bacteria, yeast or mycoplasma found.
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
no interference

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if Viability measured after exposure time points (t=3 and 60 minutes) < 50% after 3 min exposure
- The test substance is considered to be corrosive to skin if Viability measured after exposure time points (t=3 and 60 minutes) ≥ 50% after 3 min exposure AND < 15% after 60 min exposure
- The test substance is considered to be non-corrosive to skin if Viability measured after exposure time points (t=3 and 60 minutes) ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicable

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

CONTROLS
In the same experimental conditions, a positive control (50 μL of 8N KOH – Sigma, Batch No. SLBD3295V) and a negative control (50 μL of distilled water– VWR, Batch No. 1007123) were carried out. To ensure a good contact with the epidermis during all the treatment period, the liquid controls were recovered with a nylon mesh provided by Episkin SA.

Duration of treatment / exposure:

The test item was applied, at the dose of 25 mg, during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2., to the epidermal surface of 2 living human skin models previously moistened with 25 μL of distilled water.

Duration of post-treatment incubation (if applicable):

The direct interaction of MTT with the test item was checked by adding 16 mg of Copper diethylenetriamine sulfate to 300 μL of solution of MTT at 1 mg/mL. A yellow solution with the test item at the bottom of the well was observed after 3 hours of incubation between 36.3°C and 37.4°C, 5% CO2.

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 1

Value:

97.28

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 2

Value:

95.59

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 97.28 % and 95.59%, versus 91.65% and 0.31%, respectively, with the positive control item (potassium hydroxide 8N).

The mean viability of epidermises treated with the positive control during 3 minutes is 91.65%, which is not in the range of our historical data (values between 5.13% and 42.55%).

However, considering the results obtained after a treatment during 1 hour, there is no doubt that the test item is not corrosive. Therefore, it 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 results obtained under these experimental conditions enable to conclude that the test item Copper diethylenetriamine sulfate does not have to be classified in Category 1 “Corrosive”.
The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

Executive summary:

The aim of the study was to evaluate the possible corrosive effects of the test item after topical administration onin vitrohuman reconstituted epidermis (epiCS®, CellSystems®).

The test item Copper diethylenetriamine sulfate was applied as supplied, during 3 minutes and 1 hour, at the dose of 25 mg, to 2 living Human skin model surfaces (epiCS®, CellSystems®) previously moistened with 25 μL of distilled water. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The experimental protocol was established in accordance with theO.E.C.D. Test Guideline No. 431 dated 28 July 2015 and the method B.40bis of the Council regulation No. 440/2008.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 97.28 %and 95.59%, versus 91.65% and 0.31%, respectively, with the positive control item (potassium hydroxide 8N).

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item Copper diethylenetriamine sulfate does not have tobe classified in Category 1 “Corrosive”.
The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
See the documentation for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation for the category approach.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin SA, RHE/S/17
- Tissue batch number(s): Batch No. 17-RHE-124
- Production date: 11 December 2017
- Shipping date: 11 December 2017
- Delivery date: 11 December 2017
- Date of initiation of testing: 11 December 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 2780917).
- Observable damage in the tissue due to washing: The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues.
- Modifications to validated SOP: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: CV= 6.8%
- Barrier function: not determined, 6.5 cell layers
- Morphology: no abnormalities, well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: on blood of the same donor, the absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HB sp was confirmed. On epidermal cells of the smae donor, th absence of mycoplasma was confirmed.
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
no MTT interference

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)

- The test substance is considered to be non-corrosive to skin if if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
The test item is identified as requiring classification and labelling according to UN GHS (Category 2):
if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1):
if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicalble

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
The test item was applied as supplied, during 42 minutes at room temperature, at the dose of 16 mg to the epidermal surface of 3 living human skin models previously moistened with 10 μL of distilled water.



POSITIVE/NEGATIVE CONTROL
In the same experimental conditions, a positive control (5% SDS), and a negative control (DPBS – Dutscher - Batch No. 2780917) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBG6142V) in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment.
To ensure a good contact with the epidermises, during all the treatment period, the control items were recovered with a nylon mesh provided by Episkin SA.

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 hours 55 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

41.1

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean percent viability of the treated tissues was 41.1% versus 1.8% in the positive control (5% Sodium Dodecyl Sulfate).

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In accordance with the Regulation EC No. 1272/2008 and the information obtained on skin corrosion test HSMC-PH-17/0640, the test item Copper diethylenetriamine sulfate has to be classified in Category 2 “Irritating to skin”.The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.

Executive summary:

The aim was to evaluate the possible irritating effects of the test itemCopper diethylenetriamine sulfateafter topical application onin vitrohuman reconstructed epidermis (SkinEthic RHE®model).

The test item Copper diethylenetriamine sulfate was applied as supplied at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 41 hours and 55 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The experimental protocol was established in accordance with O.E.C.D.Test Guideline No. 439 adopted 28 July 2015 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The mean percent viability of the treated tissues was41.1%versus 1.8% in the positive control (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation EC No. 1272/2008 and the information obtained on skin corrosion test HSMC-PH-17/0640, the test item Copper diethylenetriamine sulfate has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
See the documentation for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation for the category approach.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

October 2nd, 2012.

Deviations:

yes

Remarks:

Deviations are considered as without impact on the conclusion of the study.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Hypharm (F-49450 La Renaudiere)
- Age at study initiation: 13 or 14 weeks
- Weight at study initiation: 2.46 - 3.10 kg
- Housing:
Each animal was kept in an individual box installed in conventional air conditioned animal husbanding:
- Diet (e.g. ad libitum) and water:
Drinking water (tap-water from public distribution system) and foodstuff (ENVIGO – 2930C) were supplied ad libitum.
Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
The temperature and relative humidity of the main test were controlled to remain within target ranges of 17°C to 23°C and 30% to 70%, respectively.
The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:

0.1 g of the test item was instilled, as supplied, into the conjunctival sac of one eye after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the test item.

Observation period (in vivo):

Ocular examinations were performed on both right and left eyes 1 hour, 24, 48 and 72 hours following treatment

Number of animals or in vitro replicates:

Three animals.
Initially, a single animal was treated. After consideration of the responses in the first animal, two additional animals were treated under the same experimental conditions.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no washing

SCORING SYSTEM:
See "any other information on materials and methods"

TOOL USED TO ASSESS SCORE: not specified

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.3

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.3

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.3

Max. score:

3

Reversibility:

not reversible

Irritation parameter:

iris score

Remarks:

lesion

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Remarks:

lesion

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 72 hours

Irritation parameter:

iris score

Remarks:

lesion

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 72 hours

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 72 hours

Irritant / corrosive response data:

The ocular reactions observed during the study have been slight to important and partially reversible:
- at the conjunctivae level: a moderate redness was noted 1 hour after the test item instillation and was totally reversible on day 7 in one animal and remaining on the last day of the test (day 21) in two animals with slight to moderate intensity (grade 1 to 2). This reaction was associated with a moderate to important chemosis noted 1 hour after the test item instillation and was totally reversible on day 2 in one animal and remaining on the last day of the test (day 21) in two animals with moderate intensity (grade 2).
- at the iris level: an injection noted 1 hour after the test item instillation in two animals and totally reversible between days 7 and 14.
- at the corneal level: a slight to moderate opacity, noted 24 hours after the test item instillation and totally reversible on day 2 in one animal and remaining on the last day of the test (day 21) in two animals with moderate intensity (grade 2).
A corneal-neovascularisation was noted between days 7 and 21.

Interpretation of results:

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

Conclusions:

The results obtained, under these experimental conditions, enable to conclude that the test item
Reaction product of copper sulfate and tetraethylenepentamine has to be classified in category 1 “irreversible effects on the eye” in accordance with the Regulation (EC) No. 1272/2008, the test item.
The signal word “Danger” and hazard statement H318 “Causes serious eye damage” are required.

Executive summary:

The test item Reaction product of copper sulfate and tetraethylenepentamine was instilled, after being reduced in fine powder, into the eye of three New Zealand rabbit at the dose of 0.1 g.
The experimental protocol was established on the basis of the official method as defined in the O.E.C.D. Test Guideline No. 405 dated October 9th, 2017.

The ocular reactions observed during the study have been slight to important and partially reversible:
- at the conjunctivae level: a moderate redness was noted 1 hour after the test item instillation and was totally reversible on day 7 in one animal and remaining on the last day of the test (day 21) in two animals with slight to moderate intensity (grade 1 to 2). This reaction was associated with a moderate to important chemosis noted 1 hour after the test item instillation and was totally reversible on day 2 in one animal and remaining on the last day of the test (day 21) in two animals with moderate intensity (grade 2).
- at the iris level: an injection noted 1 hour after the test item instillation in two animals and totally reversible between days 7 and 14.
- at the corneal level: a slight to moderate opacity, noted 24 hours after the test item instillation and totally reversible on day 2 in one animal and remaining on the last day of the test (day 21) in two animals with moderate intensity (grade 2).
A corneal-neovascularisation was noted between days 7 and 21.

In conclusion, the results obtained, under these experimental conditions, enable to conclude that the test item Reaction product of copper sulfate and tetraethylenepentamine has to be classified in category 1 “irreversible effects on the eye” in accordance with the Regulation (EC) No. 1272/2008, the test item.

The signal word “Danger” and hazard statement H318 “Causes serious eye damage” are required.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
See the documentation for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation for the category approach.

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)

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:
The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.
- Number of animals:
not specified
- Characteristics of donor animals (e.g. age, sex, weight):
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).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
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.
- Time interval prior to initiating testing:
Once all eyes had been examined and approved, the eyes were incubated between 45 and 58 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.
- indication of any existing defects or lesions in ocular tissue samples:
Eyes with (i) a fluorescein retention score > 0.5; (ii) corneal opacity > 0.5; or, (iii) any additional signs of damage were replaced.
For eyes that were not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes were rejected.
- Indication of any antibiotics used: not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg

Duration of treatment / exposure:

Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied as supplied during 10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item.
Then the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.
The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.3°C and 32.9°C.
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 45 and 58 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.
NUMBER OF REPLICATES
3
NEGATIVE CONTROL USED
Concurrent negative control (physiological saline – Dutscher Batch No. 3012808) - one replicate
POSITIVE CONTROL USED
Concurrent positive control (sodium hydroxide – Fisher Scientific, Batch No. 1550248) - three replicates

APPLICATION DOSE AND EXPOSURE TIME
Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied as supplied during 10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item.

OBSERVATION PERIOD
Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
The eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline
no

METHODS FOR MEASURED ENDPOINTS:
All observations of the cornea and measurement of corneal thickness were performed using a Haag- Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 9 1⁄2, equalling 0.095 mm.
- Corneal opacity:
Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item
- Damage to epithelium based on fluorescein retention:
The mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting:
Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. It was expressed as a percentage and was calculated from corneal thickness measurements
- Macroscopic morphological damage to the surface:
Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
- Others (e.g, histopathology): no

SCORING SYSTEM:
- Mean corneal swelling (%)
[ (corneal thickness at time t - corneal thickness at time = 0) / corneal thickness at time = 0 ] * 100
- Mean maximum opacity score
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris clearly visible
2 Easily discernible translucent area; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Irritation parameter:

cornea opacity score

Run / experiment:

Maximal mean score of corneal opacity

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean score of fluerescein retention

Value:

2.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

Maximal mean corneal swelling

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 0.3, corresponding to ICE class I;
- mean score of fluorescein retention: 2.3, corresponding to ICE class III;
- maximal mean corneal swelling: 2%, corresponding to ICE class I.
The combination of the three endpoints for the test item Reaction product of copper sulfate and tetraethylenepentamine was 1 x III, 2 x I.

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.

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 Reaction product of copper sulfate and tetraethylenepentamine 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.

Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

The test item Reaction product of copper sulfate and tetraethylenepentamine was applied as supplied, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The experimental protocol was established in accordance with theO.E.C.D. Test Guideline No. 438 adopted 26 July 2013 and the test method B.48 – Commission Regulation (EU) No. 1152/2010 dated 08 December 2010 (EU Journal L324) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The ocular reactions observed in eyes treated with the test item were:
- maximalmeanscoreofcornealopacity:0.3,correspondingtoICEclassI; - meanscoreoffluoresceinretention:2.3,correspondingtoICEclassIII;
- maximal mean corneal swelling: 2%, corresponding to ICE class I.

The combination of the three endpoints for the test item Reaction product of copper sulfate and tetraethylenepentamine was 1 x III, 2 x I.

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.

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 Reaction product of copper sulfate and tetraethylenepentamine 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 vitroand/orin vivo) are required to establish a definitive classification.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available