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Diss Factsheets

Administrative data

Description of key information

In vitro tests were performed to determine the irritation/corrosion potential of the reaction mixture of CuDTPA and CuHEEDTA. The substance was found to be irritating to both the skin and the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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-AG1428-1)
- Production date: 07 August 2017
- Shipping date: 07 August 2017
- Delivery date: 08 August 2017
- Date of initiation of testing: 08 August 201

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 3 hours between 36.2°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. 7530417).
- Observable damage in the tissue due to washing:
The rinsed tissues were checked for any coloration. The tissues applied for 3 minutes and 1 hour were noted to be slightly blue.
- 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 sample was placed in MTT solution of 1 mg/mL concentration, except the eight tissues for the non-specific control which were placed in MTT assay medium (Cell Systems Batch No. 303-AG1218), for 3 hours between 36.2°C and 37.8°C, 5% CO2..
- Spectrophotometer: not specified
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function: 55.09%
- 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
- Fresh tissues / killed tissues
As the test item was identified as potentially producing direct MTT reduction and causing colour interference, four additional killed control tissues were added to the study (two by exposition time). These four control tissues were incubated with culture medium instead of MTT solution during the MTT incubation step to generate a non-specific colour control (NSC killed).
- Procedure used to prepare the killed tissues (if applicable): freezing
- N. of replicates : 4
- Method of calculation used:
As the test item is identified as producing both direct MTT reduction and colour interference:
True viability % =
[(OD of living tissues exposed to test item - OD of killed tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT + OD of killed tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative
control] x 100

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 (8N KOH – Sigma, Batch No. SLBD3295V) and a negative control (50 μL of distilled water– Prochilab, Batch No. 20160608) were carried out.
Duration of treatment / exposure:
The test item was applied, as supplied, after being applied on a nylon mesh, at the dose of 25 mg, during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2,
Duration of post-treatment incubation (if applicable):
The skin sample was placed in MTT solution of 1 mg/mL concentration, except the eight tissues for the non-specific control which were placed in MTT assay medium (Cell Systems Batch No. 303-AG1218), for 3 hours between 36.2°C and 37.8°C, 5% CO2..
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 1
Value:
97.68
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 2
Value:
100.57
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 corrected percent viability of the epidermis skins treated with the test item were 97.68% and 100.57% (considered as 100%), versus 17.98% and 0.33%, respectively, with the positive control item (potassium hydroxide 8N).
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 Reaction mixture of CuDTPA and CuHEEDTA 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 on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

The test item Reaction mixture of CuDTPA and CuHEEDTA was applied as supplied on a nylon mesh at the dose of 25 mg then to 2 living and 4 killed Human skin model surfaces (epiCS®, CellSystems®) previously moistened with 10 μL of distilled water during 3 minutes and 1 hour. 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.

Additionally, 2 living and 4 killed Human skin model surfaces (epiCS®, CellSystems®) were treated in the same manner during both 3 minutes and 1 hour, but they were incubated in in MTT assay medium instead of MTT solution in order to generate non-specific living and killed colour controls.
The experimental protocol was established in accordance with the
O.E.C.D. Test Guideline No. 431 dated 29 July 2016 and the method B.40bis of the Council regulation No. 440/2008.

3 minutes and 1 hour after the test item application, the mean corrected percent viability of the epidermis skins treated with the test item were 97.68% and 100.57% (considered as 100%), versus 17.98% and 0.33%, 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 Reaction mixture of CuDTPA and CuHEEDTAdoes 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.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
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:
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): 17-RHE-087
- Production date: August 22, 2017
- Shipping date: August 22, 2017
- Delivery date: August 22, 2017
- Date of initiation of testing: August 22, 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. 6980417).
- Observable damage in the tissue due to washing: The rinsed tissues were checked for any coloration and noted to be slight blue and to present residue of test item.
- 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= 2.2%
- Barrier function: not determined
- 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
- Fresh tissues / killed tissues
The test item was identified as causing colour interference with the viability assay and two viable control tissues were added to the study. These control tissues were incubated with medium instead of MTT solution during the MTT incubation step to generate non-specific colour controls (a NSC living control and a NSC killed control).
As the test item was identified as producing both direct MTT reduction and colour interference two killed control tissues were added to the study, which underwent the entire testing procedure.
- Procedure used to prepare the killed tissues (if applicable): freezing
- N. of replicates : 2
- Method of calculation used:
As the test item is identified as producing both direct MTT reduction and colour interference:
True viability % = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT + OD of killed tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 100


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 directly on a nylon mesh at the dose of 16 mg then to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes at room temperature.


NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
- Concentration (if solution):

POSITIVE CONTROL
The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBF1623V) 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 epidermis, during all the treatment period, the liquid controls 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 25 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
19.9
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mean corrected percent viability of the treated tissues was 19.9%, versus 1.6% 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 with the result “non-corrosive” obtained during the skin corrosion test HSMC-PH-17/0421 carried out between 08 August 2017 and 09 August 2017, the test item Reaction mixture of CuDTPA and CuHEEDTA 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 item Reaction mixture of CuDTPA and CuHEEDTA after topical application onin vitrohuman reconstructed epidermis (SkinEthic RHE®model).

The test item Reaction mixture of CuDTPA and CuHEEDTA was applied 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 25 minutespost- 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. Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction. Moreover, 2 living and 2 killed Human skin model surfaces were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate non-specific living and killed colour controls. 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 corrected percent viability of the treated tissues was 19.9%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation EC No. 1272/2008 and with the information on skin corrosion obtained during HSMC-PH-17/0421 carried out between 08 August 2017 and 09 August 2017, the test item Reaction mixture of CuDTPA and CuHEEDTA 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

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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: 12 weeks
- Weight at study initiation: 2.01 - 2.36 kg
- Housing:
Each animal was kept in an individual box installed in conventional air conditioned animal husbanding:
- Diet (e.g. ad libitum):
Drinking water (tap-water from public distribution system) and foodstuff (ENVIGO – 2030C) were supplied ad libitum.
Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Water (e.g. ad libitum):
Drinking water (tap-water from public distribution system) and foodstuff (ENVIGO – 2030C) 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): 0.1g

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:
3
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
Max. score:
0
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
1
Reversibility:
fully reversible within: 72 h
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.3
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:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 hours
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:
0.7
Max. score:
1
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
iris score
Remarks:
lesion
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.7
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
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.3
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 totally reversible in all animals:
- at the conjunctivae level: a moderate redness noted 1 hour after the test item instillation and totally
reversible between days 2 and 7; associated with a moderate to important chemosis noted 1 hour
after the test item instillation and totally reversible between days 1 and 7.
- at the iris level: an injection noted 24 hours after the test item instillation and totally reversible on
day 3 in two animals.
- at the corneal level: a slight to moderate opacity, noted 24 hours after the test item instillation in
two animals and totally reversible between days 7 and 14.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The results obtained, under these experimental conditions, enable to conclude that the test item Reaction mixture of CuDTPA and CuHEEDTA has to be classified in category 2 “Irritating to eyes” in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.
The signal word “Warning” and hazard statement H319 “Causes serious eye irritation” are required.
Executive summary:

The test item Reaction mixture of CuDTPA and CuHEEDTA was instilled, as supplied, into the eye of three New Zealand rabbits 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 2nd, 2012.

The ocular reactions observed during the study have been slight to important and totally reversible in all animals:

  • -  at the conjunctivae level: a moderate redness noted 1 hour after the test item instillation and totally

    reversible between days 2 and 7; associated with a moderate to important chemosis noted 1 hour

    after the test item instillation and totally reversible between days 1 and 7.

  • -  at the iris level: an injection noted 24 hours after the test item instillation and totally reversible on

    day 3 in two animals.

  • -  at the corneal level: a slight to moderate opacity, noted 24 hours after the test item instillation in

    two animals and totally reversible between days 7 and 14.

    In conclusion, the results obtained, under these experimental conditions, enable to conclude that the test item Reaction mixture of CuDTPA and CuHEEDTA has to be classified in category 2 “Irritating to eyes” in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

    The signal word “Warning” and hazard statement H319 “Causes serious eye irritation” are required.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
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)
Version / remarks:
adopted 26 July 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
08 December 2010
Deviations:
no
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
- 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): see below
- Time interval prior to initiating testing:
Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 31 July 2017 at 8:25 am.
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 enucleated at Phycher on 31 July 2017 at 09:45 am.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no
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): Immediately following the zero reference measurements, three eyes (in their holder) were removed from the supervision apparatus and placed in a horizontal position. 120 mg of the test item was applied on a compress. The compress was then applied to the cornea such that the entire surface of the cornea was evenly covered with the test item.

Duration of treatment / exposure:
single application of a compress with the test item
Duration of post- treatment incubation (in vitro):
Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.
Number of animals or in vitro replicates:
three eyes
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 32.2°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, the eyes were incubated between 45 and 59 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
physiological saline – Dutscher Batch No. 3012575 - 1 replicate
SOLVENT CONTROL USED (if applicable)
not applicable
POSITIVE CONTROL USED
sodium hydroxide – Sigma, Batch No. MKBP7805V - 3 replicates
APPLICATION DOSE AND EXPOSURE TIME
During the preparation of the product, the item test became sticky making it difficult to apply to the cornea. A second preparation was therefore necessary: the test item was directly weighed on a compress and the compress was applied within 5 minutes. After the preparation 120 mg of the test item was necessary to cover the entire surface of the cornea.
Immediately following the zero reference measurements, three eyes (in their holder) were removed from the supervision apparatus and placed in a horizontal position. 120 mg of the test item was applied on a compress. The compress was then applied 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: Tthe 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.
- Indicate any deviation from test procedure in the Guideline
No deviation was registered during the study.

METHODS FOR MEASURED ENDPOINTS:
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SCORING SYSTEM:
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DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
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Irritation parameter:
cornea opacity score
Value:
<= 2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
1.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Value:
<= 13
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The combination of the three endpoints for the test item Reaction mixture of CuDTPA and CuHEEDTA was 1 x III, 2 x II.

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 2 x I, 1 x II. 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 mixture of CuDTPA and CuHEEDTA 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:

Due to its nature, the test item Reaction mixture of CuDTPA and CuHEEDTA was applied at the dose of 120 mg on a gauze in order to cover the entire surface of the cornea then 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 the O.E.C.D. Test Guideline No. 438 adopted 26 July 2013 and the test method B.48Commission 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:

  • -  maximal mean score of corneal opacity: 2.0, corresponding to ICE class III;

  • -  mean score of fluorescein retention: 1.5, corresponding to ICE class II;

  • -  maximal mean corneal swelling: 13%, corresponding to ICE class II.

    The combination of the three endpoints for the test item Reaction mixture of CuDTPA and CuHEEDTA was 1 x III, 2 x II.

    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 2 x I, 1 x II. 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 mixture of CuDTPA and CuHEEDTA 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification