Registration Dossier

Administrative data

Description of key information

Skin - Irritating
Eye - Highly irritating

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:
30-Jun-2014 to 07-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: 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)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 12.6 to 15.2 mg, moistened with 5 µl water

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 25 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:
Value:
30
Remarks on result:
other:
Remarks:
Basis: other: percentage of control. Time point: 15 minutes. (migrated information)
Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that HEDTA-H3 is irritant in the in vitro skin irritation test

Executive summary:

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with HEDTA-H3 compared to the negative control tissues was 30%. Since the mean relative tissue viability for HEDTA-H3 was below 50% after 15 minutes treatment it is considered to be irritant.

 

The positive control had a mean cell viability of 11% after 15 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 11%, indicating that the test system functioned properly
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 27 May 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:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 310.7 to 320.1 mg 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:
69
Other effects:
The corneas were turbid with a spot after the 240 minutes of treatment with HEDTA-H3. A pH effect of the test substance was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed
Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Since HEDTA-H3 induced an IVIS ≥ 55.1, it is concluded that HEDTA-H3 is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test

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 137 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.

HEDTA-H3 induced severe ocular irritation through one endpoint only (opacity), resulting in a mean in vitro irritancy score of 69 after 240 minutes of treatment

Since HEDTA-H3 induced an IVIS≥55.1, it is concluded that HEDTA-H3 is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Based on these results, the test substance should be classified as serious eye damage, category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

Findings using in vitro studies with HEDTA-H3 and non-standard methods with HEDTA-Na3 indicate that prolonged or repeated contact, particularly on abraded skin, may result in moderate irritation and burn. Short-term contact of a few minutes to hours may cause little or no apparent effects. Three studies describe corrosivity testing to US DOT standards with a 4 hour exposure, the same exposure period as used by OECD/EU test methods. HEDTA-Na3 was mixed with varying concentrations of NaOH and, at low NaOH concentration, no corrosion was noted. EDTA-Na4, a structural analogue of HEDTA, was not significantly irritating to the skin when assessed using standard methods.

 

Eye:

Eye irritation has been carried out with HEDTA-H3 and HEDTA-Na3 ex-vivo with the results indicating the potential for the undiluted substance to cause severe eye irritation. This is supported by a number of in-vivo studies with HEDTA-Na3 for the assessment of eye irritation, most of which are poorly documented. The best documented (Dow, 1982) describes a single exposure of one animal to HEDTA + 1.9% NaOH. Results indicated marked discomfort, moderate conjunctival redness and swelling, reddening of the iris and moderate corneal cloudiness. Signs of irritation were present at 21 days if not washed. The effects were considered fully reversible on washing out within 30 seconds. It is not clear the extent of the influence of NaOH on the observed irritation. Five additional, poorly documented, studies on exposure to HEDTA-Na3 products are available and indicate that exposure may cause slight to moderate eye irritation in rabbits. The information from these studies is regarded as sufficient to be able to draw conclusions regarding the need for classification and labeling.


Justification for selection of skin irritation / corrosion endpoint:
The available studies on the substance itself are in vitro studies.The available studies with the structurally related substance HEDTA-Na3 are considered suitable as supporting studies due to the non-standard methods used and limited documentation of methods and results. Results indicate that prolonged or repeated contact, particularly on abraded skin, may result in moderate irritation and burn. Short-term contact of a few minutes to hours may cause little or no apparent effects. Three studies describe corrosivity testing to US DOT standards with a 4 hour exposure, the same exposure period as used by OECD/EU test methods. HEDTA-Na3 was mixed with varying concentrations of NaOH and, at low NaOH concentration, no corrosion was noted. EDTA (viz. EDTA-Na4), a structural analogue of HEDTA, was not significantly irritating to the skin when assessed using standard methods.

Justification for selection of eye irritation endpoint:
Eye irritation has been carried out ex-vivo for both HEDTA-H3 as well as HEDTA-Na3 with the results indicating the potential for the undiluted substance to cause severe eye irritation. This is supported by a number of in-vivo studies with HEDTA-Na3 for the assessment of eye irritation, most of which are poorly documented. The best documented (Dow, 1982) describes a single exposure of one animal to HEDTA-Na3 + 1.9% NaOH. Results indicated marked discomfort, moderate conjunctival redness and swelling, reddening of the iris and moderate corneal cloudiness. Signs of irritation were present at 21 days if not washed. The effects were considered fully reversible on washing out within 30 seconds. It is not clear the extent of the influence of NaOH on the observed irritation. Five additional, poorly documented, studies on exposure to HEDTA-Na3 products are available and indicate that exposure may cause slight to moderate eye irritation in rabbits. The information from all these studies together with the information of the structurally related substance EDTA-Na4 is regarded as sufficient to be able to draw conclusions regarding the need for classification and labeling.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: highly irritating

Justification for classification or non-classification

Skin:

Based on the results of the available data the undiluted substance does require to be classified and labelled as "causes skin irritation" Category 2 - H315 according to Regulation 1272/2008/EC (CLP).

Eyes:

The information available is regarded as sufficient to be able to draw conclusions regarding the need for classification. Classification of the undiluted substance as “causes serious eye damage” Category 1 - H318 according to CLP (1272/2008/EC) is indicated. Based on the results with HEDTA-Na3, in solution at a concentration of 40% or less, a reduced classification of “causes serious eye irritation” – Category 2 - H319 according to CLP (1272/2008/EC) is indicated.