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

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

227-105-6

EC Name:

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

Cas Number:

5657-17-0

Molecular formula:

C6H12N2O4

IUPAC Name:

2-({2-[(carboxymethyl)amino]ethyl}amino)acetic acid

Test material form:

solid: particulate/powder

Details on test material:

Chemical name: Ethylenediaminediacetic acid (ED2A-H2)
Batch/lot No.: FC-C 12874
Appearance: Yellow powder (visual)
Assay: 99.2% (HPLC)
pH: 4.7 (saturated solution)
Solubility in water: soluble in water at 1.0 g/L

Specific details on test material used for the study:

Purity: 99.2%

Test system

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.

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.