2-methyldecan-1-al

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Jan 2017 - 02 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014

Deviations:

yes

Remarks:

For the MTT Assay: the formazan salt was extracted in the refrigerator instead of at room temperature. No impact on study outcome. 50µL test item used in MTT reduction/colour intereference AND test substance exposure, rather than 30µL

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-methyldecan-1-al

EC Number:

242-745-6

EC Name:

2-methyldecan-1-al

Cas Number:

19009-56-4

Molecular formula:

C11H22O

IUPAC Name:

2-methyldecanal

Test material form:

liquid

Details on test material:

Appearance: clear, colourless liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Epi-200 kits and MTT-100 assays purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes, cultured to form a multilayered, highly differentiated model of the human epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: At least 1 hour before dosing, EpiDerm™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. A 24-well plate was prepared as holding plate containing 300 µL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.
- Quality control for skin discs: Robustness of the test system (or rather of the test kit) was demonstrated following treatment with 1% Triton X-100: 4.77 hours ≤ ET50 ≤ 8.72 hours

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Rinsed 20 times
- Observable damage in the tissue due to washing: N/A
- Modifications to validated SOP: N/A

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)
- Wavelength: 570 nm
- Filter: no reference filter
- Filter bandwidth: N/A

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin as follows: < 50% mean tissue viability after 3 minutes exposure and < 15% after 60 minutes exposure results in a prediciton of corrosivity.
- The test substance is considered to be non-corrosive to skin as follows: ≥ 50% mean tissue viability after 3 minutes exposure and ≥ 15% after 60 minutes exposure results in a predition of non-corrosivity.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430: N/A

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): N/A

VEHICLE - N/A


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): N/A

Duration of treatment / exposure:

(Test item, negative and positive controls): 3 ± 0.5 minutes, 60 ± 5 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Two

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Details on study design:

TEST SITE
- Area of exposure: The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diameter).
- % coverage: Not specified
- Type of wrap if used: N/A

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a multipipette containing PBS to remove any residual test material (20 times).
- Time after start of exposure: ('At the end of exposure', no time specified)

OBSERVATION TIME POINTS
3 ± 0.5 minutes, 60 ± 5 minutes

SCORING SYSTEM:
- Method of calculation:
The mean OD of the duplicate negative control tissues was calculated after blank correction. 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 (%) = [(mean OD test item/positive control)/ (mean OD negative )]x 100

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

109.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

107.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: N/A
- Direct-MTT reduction: The colourless test item reduced MTT
Test for Direct MTT Reduction and Colour Interference:
Step 1
50 µL of the test item was added to 0.3 ml of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue and an additional test on viable tissues (without MTT addition) should be performed (step 2). Since the test item did not dye water when mixed with it, step 2 did not have to be performed.
Step 3
All test items (including those already evaluated in step 1 and step 2) should be further evaluated for their potential to interfere with MTT. To test if an item directly reduces MTT, 50 µL of the test item was added to 1 ml of a MTT/DMEM solution (1 mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. Since the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test item proved to reduce MTT and an additional test on freeze-killed tissues (step 4) was performed.
Step 4:
The procedure employed freeze-killed tissues that possess no metabolic activity but absorb and bind the test item similar to viable tissues.
The test item was applied to two freeze-killed tissues. In addition, two freeze killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues.

Data correction procedure:
True viability = Viability of treated tissue – Interference from test chemical =
ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)

- Colour interference with MTT: The colourless test item did not change colour when mixed with deionised water.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time
- Acceptance criteria met for positive control: 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
- Mean of historical values if different from the ones specified in the test guideline:
3 minutes, positive control:
Mean viability (%)= 21.60
CV (%): 9.52
Range of viabilities (%): 4.60-39.83

3 minutes, negative control:
Mean OD: 1.68
CV (OD): 4.87
Range of ODs: 1.34-1.93

60 minutes, positive control:
Mean viability (%)= 7.02
CV (%): 14.72
Range of viabilities (%): 2.80-14.77

60 minutes, negative control:
Mean OD: 1.65
CV (OD): 3.19
Range of ODs: 1.32-1.85

Any other information on results incl. tables

Table 1 details the results from all tests performed. Table 2 details the corrosivity classification based on relative mean viability.

Table 1. Results after treatment with test item and controls

DOSE GROUP	EXPOSURE INTERVAL [MIN]		AB-SORBANCE (OD) 570 NM WELL 1	AB-SORBANCE (OD) 570 NM WELL 2	AB-SORBANCE (OD) 570 NM WELL 3	MEAN AB-SORBANCE (OD) OF 3 WELLS MINUS BLANK	MEAN AB-SORBANCE (OD) OF 2 TISSUES	REL. ABSORBANCE [% OF NEGATIVE CONTROL]*	MEAN REL. ABSORBANCE [% OF NEGATIVE CONTROL]*	CV [%]	CORRECTED ODTEST ITEM**	CORRECTED REL. ABSORBANCE [% OF NEGATIVE CONTROL]***
Blank		0.046		0.041	0.035	0.000
Negative Control Tissue 1	3	1.734		1.538	1.694	1.615	1.606	100.5	100	0.8
Negative Control Tissue 2		1.659		1.624	1.631	1.597		99.5
Positive Control Tissue 1		0.497		0.479	0.472	0.442	0.473	27.5	29.4	9.2
Positive Control Tissue 2		0.540		0.547	0.546	0.503		31.3
Test Item Tissue 1		1.675		1.646	1.653	1.617	1.723	100.7	107.3	8.7	1.723- (0.164-0.199) = 1.758	109.5
Test Item Tissue 2		1.864		1.877	1.868	1.829		113.9
Negative ControlFreeze Killed TissueTissue 1		0.232		0.246	0.245	0.200	0.199
Negative ControlFreeze Killed TissueTissue 2		0.239		0.238	0.235	0.197
Test ItemFreeze Killed TissueTissue 1		0.201		0.198	0.199	0.159	0.164
Test ItemFreeze Killed TissueTissue 2		0.211		0.210	0.210	0.170
		0.035		0.035	0.034	0.000
Negative Control Tissue 1	60	1.698		1.625	1.679	1.633	1.622	100.7	100	1.0
Negative Control Tissue 2		1.668		1.648	1.617	1.610		99.3
Positive Control Tissue 1		0.158		0.163	0.161	0.126	0.140	7.8	8.7	14.5
Positive Control Tissue 2		0.195		0.192	0.182	0.155		9.5
Test Item Tissue 1		1.753		1.752	1.714	1.705	1.756	105.2	108.3	4.1	1.756- (0.182-0.166) = 1.740	107.3
Test Item Tissue 2		1.840		1.850	1.833	1.807		111.4
Negative ControlFreeze Killed TissueTissue 1		0.234		0.233	0.227	0.191	0.166
Negative ControlFreeze Killed TissueTissue 2		0.180		0.181	0.186	0.142
Test ItemFreeze Killed TissueTissue 1		0.219		0.222	0.214	0.178	0.182
Test ItemFreeze Killed TissueTissue 2		0.228		0.227	0.226	0.187

* Relative absorbance (rounded values): ( [100 x (absorbance_{test item/positive control})]/ (absorbance _{negative control} ) ) x 100

** OD_{test item} – (OD_{test item freeze-killed} – OD_{negative control freeze-killed})

*** Corrected relative viability (%) = [mean OD _{test item} - (OD _{test item freeze killed} - OD _{negative control freeze killed})/mean OD _{negative control}]x 100

Table 2. Classification of corrosivity potential based on relative viability

VIABILITY MEASURED AFTER EXPOSURE TIME POINTS	PREDICTION TO BE CONSIDERED
< 50% after 3 minutes exposure	Corrosive
≥ 50% after 3 minutes exposure AND < 15% after 60 minutes exposure	Corrosive
≥ 50% after 3 minutes exposureAND ≥ 15% after 60 minutes exposure	Non-corrosive
TEST ITEM IDENTIFIED AS CORROSIVE
< 25% after 3 minutes exposure	Optional Sub-category 1A*
≥ 25% after 3 minutes exposure	A combination of optional Sub-categories 1B and 1C

*According to the data generated in view of assessing the usefulness of the RhE test methods for supporting sub-categorisation, it was shown that around 29%, 31% and 33% of the Sub-category 1A results of the EpiDERM^TM test method may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is considered to be non-corrosive to skin since the viability after 3 minutes exposure is greater than 50% and the viability after 1 hour exposure is greater than 15%.

Executive summary:

Skin corrosion potential was measured using the Human Skin Model Test with EpiDerm™tissues models, in accordance with OECD Guideline for Testing of Chemicals 431: In vitro Skin Corrosion: Human Skin Model Test (Updated Guideline adopted July 29, 2016) and the MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014.

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Due to the test item’s MTT-reducing property, an additional test with freeze-killed tissues to determine a correction factor for true viability calculation was performed.

Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and subsequently 1 hour.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 20.5 hours in the refrigerator.

The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance compared to the negative control, both for the 3 minutes exposure period (29.4%) and for the 1 hour exposure period (8.7%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item, the corrected relative absorbance values were not reduced (109.4% after 3 minutes exposure, 107.3% after 1 hour exposure). The threshold for corrosivity defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure was not affected. Therefore, the test item is not considered to be corrosive under experimental conditions, in accordance with EU CLP and UN GHS.