4-Decenal, 9-hydroxy-5,9-dimethyl-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-17 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 439 without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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 July 22, 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

German GLP Compliance certificate (signed on 11 April 2013)

Test material

Test material information

Constituent 1

Reference substance name:

9-hydroxy-5,9-dimethyldec-4-enal

EC Number:

940-437-2

Cas Number:

926-50-1

Molecular formula:

C12H22O2

IUPAC Name:

9-hydroxy-5,9-dimethyldec-4-enal

Test material form:

liquid

Details on test material:

Appearance: colourless to pale yellow liquid
Expiry date: 18 March 2014, extended to 19 April 2014, and then 22 May 2015

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-Kit (EpiDerm™ Tissues) from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The tissues (surface of 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm of diameter).
- Tissue batch number(s): 18320
- Delivery date: 14 May 2013 (at 4°C on medium-supplemented agarose gels in a 24-well plate)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at 37ºC for 35 minutes and at RT into the sterile hood for 25 minutes.
- Temperature of post-treatment incubation (if applicable): 37 +/- 1.5°C, 5 +/- 0.5 % CO2.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and 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 three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: not reported (diluted with the MTT diluent)
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices microplate reader
- Wavelength: 570+/- 1 nm filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: 1.705, 1.994 and 1.998 (historical control equal to 1.838 with a range of viabilities between 1.423 and 2.651 ).
- Historical data and the quality certificate of the supplier of the test kit demonstrated the robustness of the test system or rather of the test kit.

MTT DIRECT INTERFERENCE
For correct interpretation of results, it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 30 µL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
> Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 tissues/dose group

PREDICTION MODEL / DECISION CRITERIA
- The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%)= [OD-test item / OD-mean of negative control] * 100

For the test item and the positive control the mean relative viability +/- standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:
- For the current test, an irritation potential of a test item according to EU classification H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control:
- mean tissue viability <= 50% : irritant (I), H315 (category 2)
- mean tissue viability > 50% : non-irritant (NI)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of the undiluted test item was dispensed directly atop the EpiDerm(TM) tissue and spread to match the surface of the tissue.
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL of DPBS
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL of 5% SLS
- Concentration (if solution): 5% w/v

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 35 at 37 °C, 5% CO2 and 95% RH and then removed from the incubator and placed in sterile hood until the 60 minutues expired.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for approximately 22 hours. The inserts were then transfered to new 6-well plates for another 18 hrs post-incubation at 37 +/- 1.5°C, 5 +/- 0.5 % CO2.

Duration of post-treatment incubation (if applicable):

- At the end of the 42 h post-treatment incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h at 37 °C, 5 % CO2 in air.
- At the end of the formazan extraction period: At the end of the formazan extraction period, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Por each tissue, 3 x 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices) with a 570 +/- 1 nm filter. Mean values were calculated from the 3 wells per tissue.

Number of replicates:

Triplicate tissues for test item, negative and positive controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure period and 42 h post-exposure incubation period

Value:

7.9

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:

- Direct MTT Reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

- Test item: The relative mean viability of the test item treated tissues was 7.9% after a 60 minutes exposure period and 42 hours post-exposure incubation period.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.899 and the standard deviation value of the percentage viability was 8.8%. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 12.8%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 10.7%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 7.3.1/1: Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item after a 60-minute exposure

 

Item	OD570 of tissues	Mean OD570 of triplicate tissues	Relative individual tissue viability (%)	Relative mean viability (% of negative control)	± SD of Relative mean viability (%)
Negative Control Item	1.705	1.899	89.8	100.0	8.8
	1.994		105.0
	1.998		105.2
Positive Control Item	0.089	0.100	8.4	5.3	12.8
	0.098		8.5
	0.114		6.9
Test Item	0.159	0.151	4.7	7.9	10.7
	0.161		5.2
	0.132		6.0

SD = Standard deviation

OD = Optical density

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions of this study and based on the result of the in vitro skin corrosion study, the test item is classified as Skin irr. Category 2 (H315: Causes skin irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EpiDerm™ Tissues reconstructed human epidermis model. 

Triplicate tissues were treated with the test item for an exposure period of 60 minutes. Each 30 µL of the test item, the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue, spread to match the tissue size. At the end of the exposure period, each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading, a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

After treatment with the negative control, the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 1.0 ≤ 2.5 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. 

After treatment with the test item, the mean relative absorbance value decreased to 7.9% versus 5.3% in the positive control (5% SLS). This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

 

Under the experimental conditions of this study and in view of the supporting skin corrosion study results, the test item is classified as Skin irr. Category 2 (H315: Causes skin irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.