Geranyl formate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 - 15 Aug 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 Jul 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

(Commission Regulation (EC) No 440/2008, 1st ATP 2009)

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:

Geranyl formate

EC Number:

203-339-4

EC Name:

Geranyl formate

Cas Number:

105-86-2

Molecular formula:

C11H18O2

IUPAC Name:

3,7-dimethylocta-2,6-dien-1-yl formate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (Epi-200)

Source strain:

not specified

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (Epi-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23349
- Delivery date: 09 Aug 2016
- Date of initiation of testing: 09 Aug 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator, thereafter at room temperature for 25 min in a sterile hood
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 2.141 ± 0.124 (acceptance criteria: 1.0 - 3.0).
- 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 1% Triton X-100. The ET-50 value was determined to be 6.16 h (acceptance criteria: 4.77 - 8.72 h).
- Contamination: The cells used to produce the EpiDermTM tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues was not performed.

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is ≤ 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% aqueous solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

approx. 42.5 h

Number of replicates:

triplicates for each treatment and control group

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 min exposure

Value:

105.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not interfere with the MTT assay (no reducing capacity).
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.613, 1.738 and 1.718) was in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.5% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability values of the test substance, the positive and negative controls in the main test were < 6% (threshold of OECD 439: <18%), thus ensuring the validity of the study.

Any other information on results incl. tables

Table 2: Results after 60 min incubation time.

Test group	Absorbance at 570 nm *			Mean absorbance of 3 tissues	Rel. absorbance (%) **			Rel. SD (%)	Rel. absorbance (% of negative control)***
	Tissue 1	Tissue 2	Tissue 3		Tissue 1	Tissue 2	Tissue 3
Negative control	1.613	1.738	1.718	1.690	95.5	102.9	101.7	4.0	100.0
Positive control	0.063	0.057	0.059	0.060	3.7	3.4	3.5	5.3	3.5
Test substance	1.837	1.705	1.787	1.776	108.7	100.9	105.7	3.8	105.1

* Mean of 3 replicate wells after blank correction

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100 × (mean absorbance test item/positive control) / (mean absorbance negative control)

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 Reconstructed Human Epidermis test the test substance does not possess any skin irritating potential.

Executive summary:

The skin irritation potential of the test substance was assessed by an in vitro skin irritation test using a reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2016). After treatment with the test substance for 60 minthe tissue viability did not decrease (105.1%) compared to the negative control (threshold for classification≤50%). Therefore, the test substance is not considered to possess an skin irritant potential.