Reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and 2-[2-(2-butoxyethoxy)ethoxy]ethyl (3,6,9,12-tetraoxahexadecyl)adipate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 - 19 Jan 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 28 July 2015

Deviations:

yes

Remarks:

no historical data of positive and negative controls

GLP compliance:

yes (incl. QA statement)

Remarks:

GROlJPE lNTERMlNISTERIEL DES PRODUITS CHIMIQUES, 67, rue Barbes, 94201 Ivry-sur-Seine CEDEX, France

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and 2-[2-(2-butoxyethoxy)ethoxy]ethyl(3,6,9,12-tetraoxahexadecyl)adipate

Molecular formula:

C26H50O10 C28H54O11

IUPAC Name:

Reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and 2-[2-(2-butoxyethoxy)ethoxy]ethyl(3,6,9,12-tetraoxahexadecyl)adipate

Test animals / tissue source

Species:

human

Strain:

other: EpiOcular™

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

in duplicates for each treatment and contol group

Details on study design:

- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-212-ver2.0, supplied by MatTek Corporation), batch No. 23759
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min exposure (37 °C), 12 min post-exposure immersion (room temperature), 2 h post-exposure (37 °C)
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary. Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.
- Description of the method used to quantify MTT formazan: The absorbance at 570 nm of each well was measured with a microplate reader (model ELx800, BioTek) and the software Gen5 ELISA V1.05.11 (BioTek). No reference wavelength measurement was used.
- Acceptability criteria: 1) Negative control: OD values of the two replicates in the range> 0.8 and< 2.5; 2) Tissues treated with the positive control substance should show a mean tissue viability < 50%; 3) The difference of viability between two tissue replicates should be less than 20% (or the SD between three replicates should not exceed 18%).
- Decision criteria/Prediction model: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is identified as not requiring classification and labelling for skin irritation. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is identified as potentially requring classification and labelling for skin irritation.

Results and discussion

In vitro

Results

Irritation parameter:

other: % mean relative absorbance of 2 tissues

Run / experiment:

30 min exposure

Value:

100.32

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control: The negative control mean OD values (0.684 and 0.585) were not in the range of > 0.8 and < 2.5. Considering the results obtained, this deviation is considered to have no impact on the conclusion of the study.
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (5.04%).
- Deviations from acceptability criteria: The difference of viability between 2 tissues for the test item was 34.52%, instead of 20% as initially scheduled. Considering the results obtained, this deviation is considered to have no impact on the conclusion of the study.

Any other information on results incl. tables

Table 1: Results after 30 min incubation time

 

	Tissue	OD	Mean OD/ disc (#)	Mean OD/ product	Viability %	Meanviability %	Differenceofviability %	Conclusion
		0.758
	I	0.635	0.684		107.80
Negativecontrol		0.659		0.635		100.00	15.60
		0.558
	2	0.607	0.585		92.20
		0.590
		0.032
	3	0.032	0.032		5.04
Positivecontrol		0.033		0.032		5.04	0.00	UN GHS Category 2 or I
		0.039
	4	0.029	0.032		5.04
		0.029
		0.622
	5	0.811	0.746		117.57
Test item		0.805		0.637		100.32	34.52	No Category
		0.519
	6	0.517	0.527		83.06
		0.545

Note: the optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

viab > 60% = No Category

viab </= 60% = Irritant

 

#: :mean of 3 values (triplicate of the same extract)

OD: opticaldensity

Acceptability criteria:

- Negative control: OD values of the two replicates in the range > 0.8 and < 2.5;

- Tissues treated with the positive control substance should show a mean tissue viability < 50%;

- The difference of viability between two tissue replicates should be less than 20% (or the SD between three replicates should not exceed 18%).

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

Under the conditions of the conducted test, the test substance is non irritating towards human-derived epidermal keratinocytes in the EpiOcular™ model.