Reaction mass of 1-hydroxypentan-2-yl formate and 2-hydroxypentyl formate and pentane-1,2-diol and pentane-1,2-diyl diformate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Mar - 28 Apr 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

Jul 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-hydroxypentan-2-yl formate and 2-hydroxypentyl formate and pentane-1,2-diol and pentane-1,2-diyl diformate

EC Number:

942-424-7

Molecular formula:

not applicable (reaction mass)

IUPAC Name:

Reaction mass of 1-hydroxypentan-2-yl formate and 2-hydroxypentyl formate and pentane-1,2-diol and pentane-1,2-diyl diformate

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 applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

120 min

Number of animals or in vitro replicates:

in duplicates for each treatment and control group

Details on study design:

- RhCE tissue construct used, including batch number: EpiOcular™ tissue (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia), batch number: 23707
- Viability: The quality of the final product was assessed by an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 15.12 min.
- Contamination: The cells used to produce the EpiOcular tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation at 37 °C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. Since the MTT solution colour did not turn blue/purple, the test substance is not presumed to have reduced the MTT solution. An additional test with freeze-killed tissues did not have to be performed.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength: 570 nm
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The test substance was considered to be not irritating to the eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is >60%.
- Acceptance criteria: The results are acceptable if the negative control OD is >0.8 and <2.5 and if the mean relative viability of the positive control is below 50% of the negative control viability.
- Reference to historical data of the RhCE tissue construct: Historical control data was used to assess the validity of the test.
- Acceptable variability between tissue replicates for the test chemical, positive and negative controls: The difference of viability between the two relating tissues of a single test substance is < 20% in the same run.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Remarks:

mean value out of 2 tissues

Run / experiment:

30 min exposure

Value:

20.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER OBSERVATIONS:
The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test substance in water or isopropanol did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.289 and 1.388) was in the range of > 1.0 and < 2.6.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the mean relative absorbance compared to the negative control to 32.5%, thus the validity of the test system is ensured.

The difference of viability between the two relating tissues of a single substance is < 20% (values differ about 0.3% to 7.3%) in the same run (for positive and negative control tissues and tissues of single test substance).

Any other information on results incl. tables

Table 2. Results after 30 min incubation time

Test group	Absorbance*		Mean absorbance of 2 tissues*	Rel. absorbance (%)**		Absolute value of the difference of the rel. absorbance (%) Tissue 1 and 2	Rel. absorbance (% of negative control)
	Tissue 1	Tissue 2		Tissue 1	Tissue 2
Negative control	1.289	1.388	1.338	96.3	103.7	7.3	100.0
Positive control	0.443	0.428	0.435	33.1	32.0	1.1	32.5
Test substance	0.278	0.282	0.280	20.7	21.1	0.3	20.9

* Mean of two replicate wells after blank correction (blank = 0.036)

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:

other: Eye Irrit. Cat. 2

Remarks:

according to Regulation (EC) 1272/2008

Conclusions:

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

Executive summary:

Irritating effects were observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (20.9%).