Ethylenediamine-N,N'-di(acetic acid)

Endpoint summary

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:

July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: 99.2%

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 14.27 to 27.95 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: other: percentage viability

Value:

129

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: percentage of control; time point: 15 minutes

Mean tissue viability +/- SD (%):

negative control:     100 +/- 6.8

positive control:      5.7 +/- 2.1

test item:              129 +/- 29

The standard deviation of the viability of the test item treated tissues was 29% which is above acceptance criteria. Since all individual viabilities were >50% and each of the individual viabilities was higher than each of the negative control viabilities, the test outcome was considered valid.

Interpretation of results:

GHS criteria not met

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 ED2A-H2 is non-irritant in the in vitro skin irritation test.

Executive summary:

The objective of this study was to evaluate ED2A-H2 for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)). The possible skin irritation potential of ED2A-H2 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch FC-C 12874 of the test item was a yellow powder. Skin tissue was moistened with 5 µl of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-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.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with ED2A-H2 compared to the negative control tissues was 129%. Since the mean relative tissue viability for ED2A-H2 was above 50% after 15 ± 0.5 minutes treatment ED2A-H2 is considered to be non-irritant.

The positive control had a mean cell viability of 5.7% after 15 ± 0.5 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 < 7% for the negative and positive control, indicating that the test system functioned properly.

In conclusion, ED2A-H2 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report andshould not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

May-June 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

purity: 99.2%

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

30 uL negative control
30 mg NaOH (positive control)
30 mg ED2A-H2

Duration of treatment / exposure:

10 sec, then rinsing with 20 mL saline

Duration of post- treatment incubation (in vitro):

240 min

Number of animals or in vitro replicates:

negative control: 1
positive control and ED2A-H2: 3

Details on study design:

Approximately 7 weeks old, male or female chickens (ROSS, spring chickens), body weight range approximately 1.5-2.5 kg, were used as eye donors. Heads of these animals were obtained from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature. Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment, using the criteria and scoring system (attached; see below). Fluorescein retention was only scored at approximately 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.
After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

Irritation parameter:

percent corneal swelling

Value:

1

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Value:

0.7

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Value:

0.5

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ED2A-H2 caused corneal effects leading to a “Not Classified” classification, consisting of no or very slight corneal swelling (mean of 1%), very slight or slight opacity (mean score of 0.7) and very slight fluorescein retention (mean score of 0.5).
The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.
The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
Microscopic examination of the corneas treated with ED2A-H2 revealed very slight erosion of the epithelium in one out of three corneas.

Interpretation of results:

GHS criteria not met

Conclusions:

Applying the classification criteria of the ICE, the following irritation classifications can be assigned: ED2A-H2: NC:“Not Classified” (UN-GHS and EU-CLP classifications)

Executive summary:

ED2A-H2 was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 mg of the test substance for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

ED2A-H2 caused corneal effects leading to a “Not Classified” classification, consisting of no or very slight corneal swelling (mean of 1%), very slight or slight opacity (mean score of 0.7) and very slight fluorescein retention (mean score of 0.5). Microscopic examination of the corneas revealed very slight erosion of the epithelium in one cornea.

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea did not reveal any abnormalities.

The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination of the corneas revealed severe erosion (3/3 corneas), severe necrosis (1/3 corneas) of the epithelium, disorder of stromal fibres (3/3 corneas) and endothelial necrosis (3/3 corneas).

Applying the classification criteria of the ICE, the following irritation classification can be assigned: NC:“Not Classified” (UN-GHS and EU-CLP classifications).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

An in vitro skin irritation with ED2A-H2 did not show skin irritation. The same applied to EDTA-H4, EDTA-Na2H2, EDTA-Na4, and EDTA-CaNa2. Findings using 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.

 

Eye:

An ex vivo eye irritation study with ED2A-H2 in chicken eyes did not show eye irritation. No eye irritation (sufficient for classification) was seen with EDTA-Na2H2 and EDTA-CaNa2; slight to moderate eye irritation was observed with EDTA-H4 and EDTA-Na4. Eye irritation testing has also been carried out with 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.

Justification for classification or non-classification

Skin:

Based on the results of the in vitro study supported by other data, the substance ED2A -H2 does not require to be classified and labelled as skin irritant.

Eyes:

Based on the results of the ex vivo study supported by other data, the substance ED2A -H2 does not require to be classified and labelled as eye irritant.