Reaction mass of (R*,R*)-7-methoxy-3,7-dimethyl-2-octanol and (R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-15 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 439 with a minor deviation: standard deviations between tissues replicates of negative control and test item groups were greater than the maximum acceptable variability. Considering the results obtained, this deviation was considered as without impact on the conclusion of the study.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

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

Version / remarks:

adopted 28 July 2015

Deviations:

yes

Remarks:

standard deviations between tissues replicates of negative control and test item groups were greater than the maximum acceptable variability

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 23 July 2009

Deviations:

yes

Remarks:

standard deviations between tissues replicates of negative control and test item groups were greater than the maximum acceptable variability

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 October 2015

Test material

Test material information

Constituent 1

Reference substance name:

(R*,R*)-7-methoxy-3,7-dimethyl-2-octanol

Cas Number:

87605-57-0

Molecular formula:

C11H24O2

IUPAC Name:

(R*,R*)-7-methoxy-3,7-dimethyl-2-octanol

Constituent 2

Reference substance name:

(R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

Cas Number:

87605-61-6

Molecular formula:

C11H24O2

IUPAC Name:

(R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

impurity 1

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No.: 171894
Purity: 99.7% (sum of the two main constituents)
Name of test material (as cited in study report): 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT
Physical state: colourless - slightly yellow liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 14 December 2017

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17 Batch No. 16-RHE-130) were received on 13 December 2016.
- On the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA, batch No. 16 MPE 133) during 3 hours and 25 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 μL of maintenance medium (Episkin SA, batch No. 16 MA 077).

TREATMENT
- The test item was applied, as supplied, at the dose of 16 µL, to the epidermal surface of 3 living skin models during 42 minutes at room temperature. To ensure a good contact with the epidermis, during all the treatment period, all liquid items were recovered with a nylon mesh provided by Episkin SA.
- In the same experimental conditions, a positive control (5% SDS), and a negative control (DPBS –PAN BIOTECH GmbH - Batch No. 9510916) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBF1623V) in a 10 mL volumetric flask (QS 10 mL of distilled water). Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermis, during all the treatment period, all liquid items were recovered with a nylon mesh provided by Episkin SA.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (PAN BIOTECH GmbH, Batch No. 9510916). The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues. They were incubated for a 41-hour and 8-minute post-treatment incubation- period in fresh medium at 37°C, 5% CO2. Then, the epidermis were put in contact with the MTT solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measured OD was proportional to the number of living cells. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

VIABILITY CALCULATION:
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1. The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.
- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%. In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

41-hour and 8-minute post-treatment incubation period in fresh medium at 37°C, 5% CO2

Number of replicates:

3 replicates of living human skin models

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

main test (duration of exposure: 42 minutes)

Value:

37.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution was observed after 3 hours of incubation between 38.0°C and 37.4°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colourless solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.001 which is less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore the test item was considered to be not interfering with the MTT assay and there was no need to add non-specific coloration controls to the study.

MTT VIABILITY ASSAY RESULTS
The mean percent viability of the treated tissues was 37.2%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate).

ACCEPTANCE OF RESULTS:
- The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.
- The standard deviation of the negative control group was 26.0%, instead of ≤18% as initially scheduled. The standard deviation of the test item group was 31.5%, instead of ≤18% as initially scheduled. Considering the results obtained, this deviation is considered as without impact on the conclusion of the study.

Any other information on results incl. tables

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	2.015	1.964	1.574	124.8	100.0	26.0
		1.908
		1.969
	2	1.513	1.610		102.3
		1.632
		1.685
	3	1.063	1.147		72.9
		1.190
		1.188
Positive control	1	0.027	0.027	0.023	1.7	1.5	0.5
		0.028
		0.027
	2	0.015	0.015		1.0
		0.015
		0.015
	3	0.027	0.028		1.8
		0.029
		0.028
Test item	1	1.057	1.069	0.586	67.9	37.2	31.5
		1.074
		1.076
	2	0.076	0.078		5.0
		0.081
		0.077
	3	0.598	0.610		38.8
		0.593
		0.638

#: mean of 3 values (triplicate of the same extract)

OD: optical density; measured after a 1:2 dilution of the formazan extracts in isopropanol.

 

Acceptability criteria: SD ≤ 18%;values of negative and positive controls were within the range of historical data.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In accordance with Regulation EC No. 1272/2008, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.

Executive summary:

An in vitro skin irritation test using the Reconstructed human Epidermis (SkinEthic RHE® model) was performed according to Guideline OECD 439 and in compliance with GLP to predict the acute skin irritation potential of the test substance.

The test substance was applied as supplied, at the dose of 16 µL, to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41-hour and 8-minute post-incubation period at 37°C, 5% CO₂. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent viability of the treated tissues was 37.2%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.

Therefore, in accordance with Regulation EC No. 1272/2008, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.