Reaction mass of [2,2,4-trimethyl-1-(2-methylpropanoyloxy)pentan-3-yl] benzoate and [2,2,4-trimethyl-3-(2-methylpropanoyloxy)pentyl] 2-methylpropanoate and (3-benzoyloxy-2,2,4-trimethylpentyl) benzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 March 2018 - 14 May 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

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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-benzoyloxy-2,2,4-trimethylpentyl isobutyrate

EC Number:

245-054-8

EC Name:

3-benzoyloxy-2,2,4-trimethylpentyl isobutyrate

Cas Number:

22527-63-5

Molecular formula:

C19H28O4

IUPAC Name:

[2,2,4-trimethyl-1-(2-methylpropanoyloxy)pentan-3-yl] benzoate

Constituent 2

Reference substance name:

2,2,4-trimethylpentane-1,3-diyl dibenzoate

EC Number:

268-316-3

EC Name:

2,2,4-trimethylpentane-1,3-diyl dibenzoate

Cas Number:

68052-23-3

Molecular formula:

C22H26O4

IUPAC Name:

(3-benzoyloxy-2,2,4-trimethylpentyl) benzoate

Constituent 3

Reference substance name:

1-isopropyl-2,2-dimethyltrimethylene diisobutyrate

EC Number:

229-934-9

EC Name:

1-isopropyl-2,2-dimethyltrimethylene diisobutyrate

Cas Number:

6846-50-0

Molecular formula:

C16H30O4

IUPAC Name:

[2,2,4-trimethyl-3-(2-methylpropanoyloxy)pentyl] 2-methylpropanoate

Test material form:

liquid

Details on test material:

- Density: 1.027
- Moisture content: 0.02%

Specific details on test material used for the study:

- Source and lot/batch No.of test material: V046803801
- Purity test date: 22 February 2018
- Expiration date of the lot/batch: 06 February 2019
- Storage condition of test material: room temperature, in the dark

Test animals / tissue source

Species:

other: in vitro study

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Protocol: In order to reach a definitive result for acute eye hazard classification, the EpiOcular™ Eye Irritation Test (EIT) was performed. MatTek EpiOcular™ tissue samples were treated in duplicate with the test article, negative control and positive control for 30 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using thiazolyl blue tetrazolium bromide (MTT) uptake and reduction. The absorbance of each sample was measured at 570 nm. The viability was then expressed as a percent of negative control values.
- Source: MatTek Corporation
- Cell line: The EpiOcular™ human cell construct
- Lot: 27424
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37±1°C
- Cell Culture Conditions: a humidified atmosphere of 5±1% CO2 in air

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl

Duration of treatment / exposure:

30 ± 2 minutes

Observation period (in vivo):

120 ± 15 minutes

Duration of post- treatment incubation (in vitro):

12 minutes

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
The EpiOcularTM tissues Lot 27424 kit F, were received from Mat-Tek on 10 Apr 2018 and refridgerated at 2-8 deg C. Cultures were pre-incubated at 37±1°C in a humidified atmosphere (80-100%) containing 5±0.5% CO2 for 60 min. At the end of the first pre-incubation period, the medium was replaced by 1 mL of fresh assay medium. Further, the tissues were pre-incubated 20 ± 4 hours at 37°C. No correction was made for the purity/composition of the test item. After pre-incubation tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS, then 50 μL of control and test items were applied topically onto the tissue surface and incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were used per treatment, negative and positive controls. After exposure period, each tissue was rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove any residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) for a 12 min immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were transferred to warm Assay Medium and were incubated for 120±15 minutes at 37°C.

- RhCE tissue construct used, including batch number
EpiOcular™ human cell construct Lot: 27324

- Doses of test chemical and control substances used
50 μL

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Exposure: 30 ± 2 minutes at 37.0 ± 1.0°C
Post-exposure immersion: 12 ± 2 minute at room temperature
Post-exposure incubation: 120±15 minutes at 37°C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μl of the test item was added to 1 ml MTT solution (1 mg/ml MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 μl tissue culture water was tested concurrently. If the MTT solution color turned blue / purple or if a blue/purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay. The test item did not reduce MTT.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
2

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
uQuant Plate Reader, Bio-Tek Instruments, Winooski, VT; 570nm

- Positive and negative control means and acceptance ranges based on historical data
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be >0.8 and <2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.

- Acceptable variability between tissue replicates for positive and negative controls
- Acceptable variability between tissue replicates for the test chemical
The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20%.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

The mean tissue viability (%)

Value:

94.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not observed

ACCEPTANCE OF RESULTS:
The mean OD570 of the negative control tissues was 1.986 (which met the acceptance criterion of greater than 0.8 and less than 2.5). The mean relative viability of positive control tissues was 31.2%, which met the acceptance criterion of less than 50%. The differences in viability between identically treated tissues were 0.17% to 7.63%, which met the acceptance criterion of less than 20%. All controls passed the acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate was not irritating to the eye in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as not requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

In order to reach a definitive result for acute eye hazard classification, the EpiOcular™ Eye Irritation Test (EIT) was performed. MatTek EpiOcular™ tissue samples were treated in duplicate with the test article, negative control and positive control for 30 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 31.2% after 30+-2 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 two tissues treated with the test item was less than 8%, indicating that the test system functioned properly. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30+-2 minutes treatment with the test item compared to the negative control tissues was 94.3%. Since the mean relative tissue viability for the test item was greater than 60% after 30 +- 2 minutes treatment it is considered to be non-irritant to the eye. In conclusion. The test item is identified as not requiring classification and labelling according to UN GHS (Category 2 or Category 1).