Reaction mass of citronellyl propionate and geranyl propionate and neryl propionate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 October 2020 - 23 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Geranyl propionate

EC Number:

203-344-1

EC Name:

Geranyl propionate

Cas Number:

105-90-8

Molecular formula:

C13H22-O2

IUPAC Name:

(2E)‐3,7‐dimethylocta‐2,6‐dien‐1‐yl propanoate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 20 EKIN 043 and 20 EKIN 047)

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
- Adult human donors
- Absence verified of HIV1 and 2 antibodies, hepatitis C antibodies, hepatitis B antigen HBs, bacteria, fungus and mycoplasma.
- This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Expiration date: 26 October 2020 (20 EKIN 043) and 23 November 2020 (20 EKIN 047)

TEMPERATURE USED FOR TEST SYSTEM
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, at 37.0 ± 1.0°C (actual range 35.9 - 37.3°C).
- Humid atmosphere of 80-100% (actual range 80-95%), containing 5.0 +/- 0.5% CO2 in air in the dark.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.

TEST FOR COLOR INTERFERENCE BY THE TEST ITEM
- To assess the color interference, 50 μL of the test item or 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water.
- The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark.
- 50 μL of the test item or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol.
- The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking.
- If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.

TEST FOR REDUCTION OF MTT BY THE TEST ITEM
- 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 sterile Milli-Q 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours at 37.0+/-1.0 C in the dark; negative control milli-Q water
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per test item together with negative and positive
controls.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 runs

ACCEPTABILITY CRITERIA
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and upper acceptance limit ≤1.5) and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

INTERPRETATION (See Table 1)
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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Twenty-five μL of the undiluted test item on top of the skin tissues

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37°C.

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

First experiment

Value:

49

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Second experiment

Value:

29

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

FIRST EXPERIMENT
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 49% ± 59%. Since the mean relative tissue viability for the test item was below or equal to 50% after 15 ± 0.5 minutes treatment it is considered to be irritant. However, since the individual viabilities (16%, 16% and 117%) were spread over two categories, the experiment was
repeated.

SECOND EXPERIMENT
In the repeat experiment, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 29% ± 16% (individual viabilities of 21%, 48% and 19%). Since the treated tissues had an individual cell viability after exposure to the test item below 50% and the mean relative tissue viability for the test item in the repeat experiment was well below 50% after 15 ± 0.5 minutes treatment, the test item is considered to be irritant.

ACCEPTANCE OF RESULTS
The positive control had a mean cell viability of 3.7% ± 1.1 and 5.2% ± 22.9 (experiment 1 and 2 respectively) 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 ≤ 16%, indicating that the test system functioned properly. Except for the three tissues treated with test item in experiment 1, which had a standard deviation of 59%. Therefore, the repeat experiment was performed.

Any other information on results incl. tables

Table 2. Mean Absorption in the In Vitro Skin Irritation Test with Geranyl Propionate

First experiment:

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.037	1.054	1.127	1.072	±	0.048
Test item	0.167	0.166	1.255	0.529	±	0.628
Positive control	0.047	0.027	0.046	0.040	±	0.011

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 (0.04373). Isopropanol was used to measure the background absorption.

 

Repeat experiment:

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.091	1.131	0.933	1.052	±	0.104
Test item	0.220	0.506	0.203	0.310	±	0.170
Positive control	0.037	0.038	0.090	0.055	±	0.030

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 (0.04345). Isopropanol was used to measure the background absorption.

 

Table 3. Mean Tissue Viability in the In Vitro Skin Irritation Test with Geranyl Propionate

First experiment:

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	4.4
Test item	49	59
Positive control	3.7	1.1

 

Second experiment:

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	9.9
Test item	29	16
Positive control	5.2	2.9

Applicant's summary and conclusion

Interpretation of results:

other: Skin irritant in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance causes skin irritation in the in vitro skin irritation test (OECD guideline 439).

Executive summary:

An in vitro skin irritation test according to OECD guideline 439 and in accordance with GLP principles was performed. 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. 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 49%. Since the mean relative tissue viability for the test item was below or equal to 50% after 15 ± 0.5 minutes treatment it is considered to be irritant. However, since the individual viabilities (16%, 16% and 117%) were spread over two categories, the experiment was repeated. In the repeat experiment, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 29% (individual viabilities of 21%, 48% and 19%). Since the treated tissues had an individual cell viability after exposure to the test item below 50% and the mean relative tissue viability for the test item in the repeat experiment was well below 50% after 15 ± 0.5 minutes treatment, the test item is considered to be irritant. The positive control had a mean cell viability of 3.7% and 5.2% (experiment 1 and 2 respectively) 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 ≤ 16%, indicating that the test system functioned properly. Except for the three tissues treated with test item in experiment 1, which had a standard deviation of 59%. Therefore, the repeat experiment was performed. In conclusion, the test substance causes skin irritation in the in vitro skin irritation test (OECD guideline 439).