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Description of key information

The in vitro skin irritation test showed inconclusive results with regard to skin irritation; however the in vitro skin corrosion test with the structurally related substance EDTA-Cu(NH4)2 was negative. In addition, two studies were available with EDTA-CuNa2 (see also read across document in section 13) on skin irritation, one using a 80% solution, the other study using a 50% solution. The 80% solution showed slight skin irritation but not sufficient for classification, the 50% solution did not show skin irritation.

The in vitro eye irritation test showed borderline irritation in the BCOP (score was 3.1 which just > 3). No eye irritation was seen in vitro with EDTA-Cu(NH4)2.

Two in vivo studies were also available with EDTA-CuNa2 for eye irritation. The key study with EDTA-CuNa2 showed irritation just sufficient to require classification, the supporting study showed slight irritation not sufficient for classification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-Jun-2014 to 28-Aug-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Deviations:
no
GLP compliance:
yes (incl. certificate)
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg, moistened with 5 µl water

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 10 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 10 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
Exposure:15 minutes
Post incubation period: 42 hours
Details on study design:
TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Irritation / corrosion parameter:
other: other: percentage viability
Value:
75
Remarks on result:
other:
Remarks:
Basis: other: percentage of control. Time point: 15 minutes. (migrated information)
Irritation / corrosion parameter:
other: other: percentage viability
Value:
58
Remarks on result:
other:
Remarks:
Basis: other: percentage. Time point: 15 minutes. (migrated information)

Skin irritation is expressed as the remaining cell viability after exposure to the test substance.

The relative tissue viability for EDTA-CuK2 ranged from 6 to 111% in three independent experiments. Consequently, the result of these experiments is inconclusive.

 

Interpretation of results:
other: inconclusive
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
The relative tissue viability for EDTA-CuK2 ranged from 6 to 111% in three independent experiments. Consequently, the result of these experiments is inconclusive.

Based on the results of this test it should be labelled, according to Regulation (EC) No 1272/2008 (CLP), as: Causes skin irritation (H315).
Executive summary:

Skin irritation is expressed as the remaining cell viability after exposure to the test substance.

In the initial assay (first experiment) the relative tissue viability obtained after 15 minutes treatment with EDTA-CuK2 compared to the negative control tissues was 92, 94 and 40% for the individual tissues (mean 75%). Since the SD calculated from the individual % tissues viability of the three identically treated replicates was 31%, the acceptability criteria were not met and the experiment was repeated.

In the second experiment the relative tissue viability obtained after 15 minutes treatment with EDTA-CuK2 compared to the negative control tissues was 12, 15 and 13% for the individual tissues (mean 13%). This test was rejected due to a low response (OD570) of the negative control tissues and consequently an additional experiment was performed (protocol deviation 3).

In the third experiment the relative tissue viability obtained after 15 minutes treatment with EDTA-CuK2 compared to the negative control tissues was 55, 111 and 6% for the individual tissues (mean 58%). Since the SD calculated from the individual % tissues viability of the three identically treated replicates was 53%, the acceptability criteria were not met.

The relative tissue viability for EDTA-CuK2 ranged from 6 to 111% in three independent experiments. Consequently, the result of these experiments is inconclusive.

The positive control had a mean cell viability of 35, 21 and 40% after 15 minutes exposure in the three subsequently performed experiments, respectively. The absolute mean OD570(optical density at 570 nm) of the negative control tissues in the first and third experiment was within the laboratory historical control data range. The standard deviation of the percentage viability of three tissues treated identically with reference substances was less than 20%.

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:
26-May-2014 to 01-Jul-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
other: OECD guideline 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage”
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants
Deviations:
no
GLP compliance:
yes (incl. certificate)
Vehicle:
physiological saline
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20% (w/w) solution (750 µl) per cornea

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole


Duration of treatment / exposure:
240 minutes
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 240 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed by immediate opacity measurement and permeability evaluation of the cornea.

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
In vitro score range --> UN GHS
≤ 3 --> No Category
> 3; ≤ 55 --> No prediction can be made
>55 --> Category 1

Irritation parameter:
other: In vitro irritancy score (IVIS)
Basis:
mean
Time point:
other: 240 minutes
Score:
4
Irritation parameter:
other: In vitro irritancy score (IVIS)
Basis:
mean
Time point:
other: 240 minutes
Score:
3.1
Other effects:
The corneas were translucent after the 240 minutes of treatment.
Interpretation of results:
slightly irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: based on IVIS > 3 but ≤ 55.1
Conclusions:
EDTA-CuK2 induced an IVIS > 3 but ≤ 55.1 which means borderline irritating; no prediction on the classification can be made based on the outcome of this study alone.
Executive summary:

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was EDTA-CuK2 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

EDTA-CuK2 showed in vitro irritancy scores of 6.0, 0.0 and 4.9 after 240 minutes of treatment,resulting in a mean in vitro irritancy score of 4.0 after 240 minutes of treatment. Since one out of three corneas showed an IVIS below 3, the test was repeated, resulting in IVIS scores of -4.1, -0.1 and 3.1, resulting in a mean in vitro irritancy score of 3.1 after 240 minutes of treatment.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Most metal chelates are not irritating to the eyes (see read across document in section 13). However, EDTA-CuK2, like EDTA-CuNa2 showed borderline eye irritation, just requiring classification and labeling. Most metal chelates are neither irritating to the skin; however, EDTA-CuK2 showed inconclusive results in vitro. Based on read across with EDTA-Cu(NH4)2 which was irritating but not corrosive to the skin in vitro, it was concluded that also EDTA-CuK2 was irritating to the skin.

Justification for selection of skin irritation / corrosion endpoint:

The in vitro skin irritation study with EDTA-CuK2 showed inconclusive results (3 repeat experiments). Based on read across with EDTA-Cu(NH4)2 it was concluded that also EDTA-CuK2 was considered to be a skin irritant.

Justification for selection of eye irritation endpoint:

Relatively well performed and reported study in vitro study that showed borderline eye irritation. Based on read across with EDTA-CuNa2 it was concluded that also EDTA-CuK2 was considered to be an eye irritant.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Based on the results of the in vitro skin irritation study and read across with EDTA-CuNa2 and EDTA-Cu(NH4)2, it was concluded that no classification was needed for corrosion but for irritation. For the endpoint skin irritation classification with GHS Cat. 2 should be applied based on the skin irritation observed. Based on the in vitro eye irritation test and the in vivo results with EDTA-CuNa2, classification GHS Cat. 2 is required for eye irritation.