Sodium (2-butoxyethoxy)acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 25, 2018 - May 29, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

28 April 2017

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Version / remarks:

Version 1.8 (February 2009)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium (2-butoxyethoxy)acetate

EC Number:

268-040-3

EC Name:

Sodium (2-butoxyethoxy)acetate

Cas Number:

67990-17-4

Molecular formula:

C8H16O4.Na

IUPAC Name:

sodium (2-butoxyethoxy)acetate

Test material form:

liquid: viscous

Details on test material:

Batch Number: P59564
Appearance: white-yellow, low melting point paste
Purity: 100 %
Expiration date: 31.12.2018
Storage: room temperature (ca. 20°C)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult human donors

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin(TM) Small Model, a three-dimensional human epidermis model
- Tissue batch number(s): SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 – France

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: approximately 25 mL PBS 1 x solution; rest of the PBS was removed from epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis)
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL stock solution; 2 mL of 0.3 mg/mL per well
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50% of the negative control.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50% of the negaive control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5% aq. solution

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1st run

Value:

72

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

24%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: yes
During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary. The non-specific MTT reduction (NSMTT) was determined to be 0.486 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colouring potential of test item:
The test item showed no ability to become coloured in contact with water. However, the test item has an intrinsic colour (white-yellow), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.031. The Non Specific Colour % (NSC %) was calculated as 4.7 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1: OD values and viability percentages of the controls:

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.604	91
	2	0.761	115
	3	0.621	94
	mean	0.662	100
	standard deviation (SD)		13.04
Positive Control: SDS (5 % aq.)	1	0.143	22
	2	0.236	36
	3	0.098	15
	mean	0.159	24
	standard deviation (SD)		10.65

Table 2: OD values and viability percentages of the test item:

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
sodium (2-butoxyethoxy) acetate	1	0.483	0.480	73	72
	2	0.451	0.448	68	68
	3	0.502	0.499	76	75
	mean	0.479	0.476	72	72
	standard deviation (SD)			3.93	3.93

Table 3: OD values of additional controls for MTT-interacting test item:

Additional controls	Optical Density (OD)
Negative control killed tissues: 1x PBS	1	0.065
	2	0.084
	3	0.055
	mean	0.068
Test item treated killed tissues: sodium (2-butoxyethoxy) acetate	1	0.082
	2	0.068
	3	0.063
	mean	0.071

Table 4: OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSC %)
sodium (2-butoxyethoxy) acetate (test item treated tissueswithout MTT incubation)	1	0.029	4.7
	2	0.034
	mean	0.031

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item sodium (2-butoxyethoxy) acetate is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

A study according OECD TG 439 was conducted to determine the skin irritation potential of the test item. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours (± 1h) in an incubator with 5±1% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1°C in 5±1% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5% aq.) and 1xPBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. The test item is a MTT-reducer and has an intrinsic colour (white-yellow). To avoid a possible double correction for colour interference, a third control for non-specific colour in killed tissues was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference. The test item showed no ability to become coloured in contact with water. However, the test item has an intrinsic colour (white-yellow), two additional test item-treated tissues were used for the non-specific OD evaluation.

In this in vitro skin irritation test using the EPISKIN model, the test item sodium (2-butoxyethoxy) acetate did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 72 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.