Reaction mass of [(3,7-dimethyloct-6-en-1-yl)oxy]acetaldehyde and [(3,7-dimethyloctyl)oxy]acetaldehyde

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February 2018 - 23 February 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

Reaction mass of [(3,7-dimethyloct-6-en-1-yl)oxy]acetaldehyde and [(3,7-dimethyloctyl)oxy]acetaldehyde

EC Number:

913-400-3

Molecular formula:

C12H22O2

IUPAC Name:

Reaction mass of [(3,7-dimethyloct-6-en-1-yl)oxy]acetaldehyde and [(3,7-dimethyloctyl)oxy]acetaldehyde

Test material form:

liquid

Details on test material:

Storage condition of test material: PURGE HEADSPACE WITH NITROGEN, REFRIGERATE (35 - 46.5 F / 2 - 8 C)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissue
- Tissue batch number(s): 27958

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes exposure and 60 minute exposure: 37 ± 1.0 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. After 1 hour of treatment the test item could not be removed completely.
- Observable damage in the tissue due to washing: no

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
The test substance 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 or 50 μL Milli-Q water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.
The test substance 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 1-hour exposure. To assess the color interference, 50 μL of the test item or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Microplate reader: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

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

EVALUATION
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

PREDICTION MODEL / DECISION CRITERIA: see Table 1

ACCEPTABILITY CRITERIA:
1. The absolute mean OD570 of the two tissues of the negative control should reasonably be >= 0.8 and <= 2.8 and be within the laboratory historical control data range.
2. The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
3. In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be <= 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL

Duration of treatment / exposure:

3 minutes and 60 minutes

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure / mean of 2 replicates

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

7.5

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure / mean of 2 replicates

Value:

89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

6.0

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.723 and 1.862)
- Acceptance criteria met for positive control: yes, the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 17%

Any other information on results incl. tables

Mean Absorption in the in vitro Skin Corrosion Test:

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.813	1.633	1.723	±	0.127	1.778	1.945	1.862	±	0.118
Muguet Aldehyde	1.722	1.641	1.682	±	0.058	1.510	1.815	1.662	±	0.216
Positive control	0.139	0.119	0.129	±	0.014	0.118	0.104	0.111	±	0.010

SD = Standard deviation

Duplicate exposures are indicated by A and B.

Mean Tissue Viability in the in vitro Skin Corrosion Test:

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Muguet Aldehyde	98	89
Positive control	7.5	6.0

Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	9.9	8.6
Muguet Aldehyde	4.8	17
Positive control	14	12

CV (%) = 100 - [(lowest OD₅₇₀/highest OD₅₇₀) x 100%]

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive to the skin

Remarks:

In accordance with Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The results of an in vitro skin corrosion test showed that the substance was not corrosive to the skin.

Executive summary:

The substance was tested in duplicate in an in vitro skin corrosion test according to OECD TG 431 test guideline and GLP principles. Tissues were exposed to the substance, a negative control (Milli-Q water) and a positive control (8.0 N KOH) for 3 minutes and 60 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative control, positive control and variability between measurements were met.

The cell viability of the tissues exposed to the substance were 98% and 89% for 3 minutes and 60 minutes exposure, respectively. Both values did not exceed thereshold for corrosivity (50% after 3 minutes exposure and 15% after 60 minutes exposure), therefore the substance is considered not to be corrosive.