N,N-bis(2-hydroxyethyl)glycine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 January 2017 - 27 February 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:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-hydroxyethyl)glycine

EC Number:

205-755-1

EC Name:

N,N-bis(2-hydroxyethyl)glycine

Cas Number:

150-25-4

Molecular formula:

C6H13NO4

IUPAC Name:

N,N-bis(2-hydroxyethyl)glycine

impurity 1

Reference substance name:

N-(2-hydroxyethyl)ethylenediaminetriacetic acid

EC Number:

205-759-3

EC Name:

N-(2-hydroxyethyl)ethylenediaminetriacetic acid

Cas Number:

150-39-0

Molecular formula:

C10H18N2O7

IUPAC Name:

N-{2-[bis(carboxymethyl)amino]ethyl}-N-(2-hydroxyethyl)glycine

impurity 2

Reference substance name:

Not specified

IUPAC Name:

Not specified

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

solid: crystalline

Details on test material:

Appearance: white crystalline powder
Storage conditions: at room temperature protected from light

Specific details on test material used for the study:

Stable at higher temperatures: Yes, with a maximum temperature of 40 °C and for a maximum duration of 2 days.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 22 hours at 37°C

ENVIRONMENTAL CONDITIONS
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.0-37.1 °C
- Humidity (%): 64-87

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1, with phosphate buffered saline
- Observable damage in the tissue due to washing: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is = 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

TEST ITEM:
18.9 to 32.0 mg on skin moistened with 5 µl Milli-Q water

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate
- Re-spread after 7 minutes contact time

Duration of treatment / exposure:

15 ±0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Test system

Details on study design:

TEST SITE
- EPISKIN Standard Model (TM) (EPISKIN-SM (TM), 0.38 cm^2, Lot no.: 17-EKIN-008).This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days , which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

SCORING SYSTEM:
- 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.
- The test item was checked for possible direct MTT reduction and colour interference

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(100%)

Positive controls validity:

valid

Remarks:

(22%)

Remarks on result:

other: SD of test item: 2.6%

Other effects / acceptance of results:

- The positive control had a mean cell viability of 22% after 15 minutes of exposure. The absolute mean OD570 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 6%, indicating that the test system functioned properly.
- Since all acceptability criteria are met, the study is considered to be valid.

Any other information on results incl. tables

The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. No colour change was observed, therefore it was concluded that the test substance did not interact with MTT.

The mean optical densities at 570 nm (OD570) were:

Negative control: 0.835 ±0.033

Test item: 0.784 ±0.021

Positive control: 0.186 ±0.043

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

(CLP criteria also not met)

Conclusions:

An in vitro skin irritation test with Bicin was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the substance is not irritating in the in vitro skin irritation test according to the GHS and CLP criteria, under the experimental conditions described in this report.