dec-1-en-4-yne

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30-05-2017 to 30-06-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

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)

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2015 ; signature: September 2015

Test material

Test material information

Constituent 1

Reference substance name:

dec-1-en-4-yne

EC Number:

813-331-8

Cas Number:

24948-66-1

Molecular formula:

C10H16

IUPAC Name:

dec-1-en-4-yne

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: At 2-8 °C in the refrigerator with nitrogen cover gas
- Other: Slightly yellow

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN TM Small Model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM TM Epi-200- SIT
- Tissue batch number(s): Lot no.: 25825
- Production date: Not reported.
- Shipping date: Not reported.
- Delivery date: 27-06-2017 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 27-05-2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C, 5 ± 0.5% CO2 ; Tissues were incubated for nearly 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation medium will be changed (0.9 mL of pre-warmed fresh medium). Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was approximately 41 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 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 item. 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. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate for incubation.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution prepared in assay medium
- Incubation time: The plate was post-42 hour incubation period – further incubated (37 ± 1.5 °C, 5 ± 0.5% CO2) for 3 hours for the MTT assay.
- Spectrophotometer: microplate reader
- Wavelength: 570 nm (OD570)
- Filter: Without reference filter
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer: Not reported.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control following the 60-minute exposure/42-hour incubation period. With standard deviation of 0.6%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 1.670 and the standard deviation of viability was 4.6%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 7.0%. The mean OD570 for both the negative and positive controls were within the historical control ranges.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.

NUMBER OF REPLICATE TISSUES: Three (3), triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In the pretest for colour interference: the solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues. In the pre-test for direct MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. It was unnecessary to run killed tissues.
- Procedure used to prepare the killed tissues (if applicable): Not applicable.
- N. of replicates : Not applicable.
- Method of calculation used: Not applicable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if e.g. the viability after 60-minutes exposure / 42-hours incubation is less than or equal to 50%,.
- The test substance is considered to be non-irritating to skin if e.g. the viability after 60-minutes exposure / 42-hours incubation is greater than 50%.
- Cut off points in accordance with OECD TG 439 and the GHS and CLP Classification systems.
- Skin irritation is expressed as the remaining cell viability after exposure to the test substance at exposure times 60-minutes / 42-hours incubation. Where necessary, direct MTT reduction, colour interference was completed.

OTHER:
Epi-200- SIT Model (Lot no.: 25825). 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 multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅). The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential. As a complimentary endpoint the concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that 1. air bubbles between agarose and insert were not > 30% of the total surface, 2. liquid on top of the insert was removed with sterile cotton tips, 3. if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded. 0.9 mL of the assay medium (20 – 25 °C) were pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further about 22 hours (37 ± 1.5 °C, 5 ± 0.5% CO2, 95 ± 5% RH).

Application of test item and rinsing:
30 μL (47 μL/cm2 according to guideline) of the undiluted test item was dispensed directly atop the EpiDerm™ tissue and spread to match the surface of the tissue for a complete treatment time of 60 minutes. The test was performed under monochromatic yellow light. 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 item. 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. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate for incubation. Tissues were incubated for nearly 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The tissues were subsequently placed into was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium (and sample stored in the freezer). After incubation medium will be changed (0.9 mL of pre-warmed fresh medium). Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was approximately 41 hours. Following ca. 42 hour post exposure incubation the treated plates were then tested for MTT formazan extraction.

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
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl ; Dulbecco’s Phosphate Buffered Saline (DPBS)
- Concentration (if solution): Tested as supplied (undiluted).

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl ; The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution.
- Concentration (if solution): 5%

Duration of treatment / exposure:

Tissues were treated with the test item for 60 minutes and then washed with DPBS to remove residual test item. Negative control and/or Positive control were similarly treated with the respective reference items (DPBS and/or SDS 5%).

Duration of post-treatment incubation (if applicable):

Subsequently the skin tissues were incubated for 42 hours at 37°C. Then 3 hours incubation prior to and/or MTT loading and Formazan extraction.

Number of replicates:

Triplicate (n=3) ; treatment and concurrent negative control and positive control groups

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Value:

87.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 minute exposure. Reversibility: no data. Remarks: n=3; SD = 7.0% ; Score in terms of percentage of negative control.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has previously demonstrated technical proficiency. Also see historic control data.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data.

Any other information on results incl. tables

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item

Dose Group	Tissue No.	Absorbance 570 nm Well 1	Absorbance 570 nm Well 2	Absorbance 570 nm Well 3	Mean Absorbance of 3 Wells	Mean Absorbance of three Wells blank corrected	Mean Absorbance of 3 Tissues	Rel. Absorbance [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]	Mean Rel. Absorbance [%] or “Mean viability”
Blank		0.038	0.038	0.037	0.037	0.000
Negative Control	1	1.708	1.718	1.740	1.722	1.684	1.670	100.8	4.6	100.0
	2	1.804	1.757	1.767	1.776	1.738		104.1
	3	1.659	1.606	1.611	1.625	1.588		95.1
Positive Control	1	0.090	0.095	0.094	0.093	0.055	0.055	3.3	0.6	3.3
	2	0.093	0.093	0.091	0.092	0.055		3.3
	3	0.092	0.093	0.093	0.093	0.055		3.3
Test Item	1	1.406	1.385	1.369	1.387	1.349	1.464	80.8	7.0	87.6
	2	1.552	1.532	1.523	1.536	1.498		89.7
	3	1.590	1.562	1.593	1.582	1.544		92.5

 

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues (mean OD: between 1.625 and 1.776). Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.3% (= ≤ 20%) thus ensuring the validity of the test system. The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were 7%.All assay acceptance criteria were met.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this in vitro study, the test item is not considered to be irritating to skin.

Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 under GLP to assess the skin irritation potential of the test item using a human three dimensional epidermal model (Epi-200- SIT / EPIDERM Model). Triplicate tissues were treated with the test item, negative control and positive control items for an exposure period of 60 minutes. The test item, the negative control (DPBS) and of the positive control (5% SLS) were applied to each triplicate tissue, and spread to match the surface of triplicate tissue. At the end of the exposure period each tissue was rinsed before incubating for ca. 42 hours. After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1.5 °C, 5 ± 0.5 % CO2), the tissues were rinsed three times with DPBS, and carefully dried with blotting paper. The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues are completely covered. The 24-well plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for 2.5 hours while shaking at room temperature. After the extraction period was completed, 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. Per each tissue, three individual 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. 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). The relative mean viability of the test item treated tissues was 87.6% after the 60-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 7.0%. The mean OD570 for the negative control treated tissues was 1.670 and the standard deviation value of the viability was 4.6%. The absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6%. The positive control acceptance criteria were therefore satisfied. All acceptance criteria were considered to be met. Under the conditions of this study, the test item is not considered to be irritating to the skin.