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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27.06.2018-01.10.2018

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

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:

Batch (Lot) Number: V046812101
Expiry date: Clear colourless liquid
Storage Conditions: At room temperature
Test Facility test item number: 209637/A
Test item handling: No specific handling conditions required

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize 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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model TM (SkinEthic Laboratories, Lyon, France).
- Tissue batch number(s): EPISKIN-SMTM, 0.38 cm2, Batch no.: 18 EKIN 035
- Production date: August 28, 2018
- Date of initiation of testing: August 28, 2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml MTT solution (0.3 mg/ml in PBS)
- Incubation time: 3h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μl
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5% SDS in PBS

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

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): 25 μl
- Concentration (if solution): 100% (undiluted)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5% SDS in PBS

Duration of treatment / exposure:

15 ± 0.5 minutes

Observation period:

42 hours

Number of animals:

not used, in vitro study

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the tissues were washed with phosphate buffered saline to remove residual test item
- Time after start of exposure: 15 ± 0.5 minutes

OBSERVATION TIME POINTS
The skin tissues were incubated for 42 hours at 37°C. After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 µL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69 hours.

SCORING SYSTEM:

A test item is considered irritant in the skin irritation test 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.
A test item is considered non-irritant in the in vitro skin irritation test 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.

Data interpretation of test items

Relative mean viability of 3 individual tissues after 15 minutes of exposure and 42 hours of post incubation Prediction to be considered

≤ 50% of the mean viability of the negative controls Category 1 or Category 2
(additional information on corrosion needed)

> 50% of the mean viability of the negative controls No category

Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

105

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: 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, at least 25 μl of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed. No color change were observed.
- Colour interference with MTT: The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, 10 μl of the test item was added to 90 μl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μl Milli-Q water was tested concurrently. At the end of the shaking period a color check was performed. No color changes were observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 105%. The absolute mean OD570(optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range.
- Acceptance criteria met for positive control: the positive control had a mean cell viability of 26% after 15 ± 0.5 minutes exposure. The mean relative tissue viability of the positive control should be <=40% relative to the negative control.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 8%. The SD calculated from individual % tissue viabilities of the three identically treated replicates should be <=18.

Any other information on results incl. tables

Mean Absorption in the In Vitro Skin Irritation Test

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.012	1.109	1.170	1.097	±	0.079
Test substance	1.218	1.168	1.082	1.156	±	0.069
Positive control	0.288	0.327	0.253	0.290	±	0.037

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.0422). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in the In VitroSkin Irritation Test

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	7.2
Test substance	105	6.3
Positive control	26	3.4

	Negative control (absorption; OD570)	Positive control (absorption; OD570)
Range	0.422 – 1.547	0.023 – 0.437
Mean	0.98	0.13
SD	0.18	0.08
n	174	173

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2014 to November 2017.

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 is non-irritant in the in vitro skin irritation test under the experimental conditions described and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The objective of this study was to evaluate the test substance for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item was applied undiluted (25 µl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive control had a mean cell viability of 26% after 15 ± 0.5 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 three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 105%. Since the individual values were above 50% test item is considered to be non-irritant.

In conclusion, the test substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report andshould not be classified.