2-(3,3-dimethylcyclohex-1-enyl)-2,5,5-trimethyl-1,3-dioxane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July - 14 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study performed according to OECD Guideline 439.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

26 July 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24 August 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

08 April 2015.

Test material

Test material information

Constituent 1

Reference substance name:

2-(3,3-dimethylcyclohex-1-en-1-yl)-2,5,5-trimethyl-1,3-dioxane

EC Number:

815-500-1

Cas Number:

1853175-99-1

Molecular formula:

C15 H26 O2

IUPAC Name:

2-(3,3-dimethylcyclohex-1-en-1-yl)-2,5,5-trimethyl-1,3-dioxane

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: room temperature, darkness.

In vitro test system

Test system:

human skin model

Remarks:

Epi-200 Kit

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE:
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava.
Date received: 14 August 2015
EpiSkinTM Tissues (0.38cm2) lot number: 21685
Maintenance Medium lot number: 1660120
Assay Medium lot number: 080615ZSD

EVALUATION OF DIRECT INTERACTION WITH MTT
- For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. The direct interaction of MTT with the test item was checked by adding 30 µL of the test item to 1mL of the solution of MTT. No colour change could be observed in the present study. Therefore, there is no direct interaction between the test item and MTT

TREATMENT
- After approximately 18 hours incubation of the tissues, they were treated with the test item.
- 1 plate (3 tissues) was used as negative control; each tissue was treated with 30 μL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue sur-face.
- 1 plate was used as positive control; each tissue was treated with 30 μL SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- 1 plate was used for treatment with the test item: 30 μL test item were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in 1-min-intervals. After dosing the last tissue, all plates are trans-ferred into the incubator for 35 min at 37 ± 1°C and 5.0 ± 0.5% CO2. 60 min after the first application, the inserts were removed from the plates using sterile forceps and rinsed im-mediately in 1-min-intervals. After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). Then, the tissues were set in the incubator for 24 h.

REMOVAL OF TEST MATERIAL AND CONTROLS
- For 3 incubated tissues, a new 6-well-plate with 0.9 mL assay medium in the upper row was prepared. The tissues were removed from the incubator and shaken for 10 min (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the in-cubator for 18 h for post-incubation.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE

- After a total incubation time of 42 h, a 24-well-plate was prepared with 300 μL freshly pre-pared MTT-reagent in each well. The tissues were blotted on the bottom and then trans-ferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 h at 37 ± 1°C and 5.0 ± 0.5% CO2.
- After 2 h, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
- From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.

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

ACCEPTABILITY OF THE ASSAY
- The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the two tissues is ≥ 0.8 and <=2.8.
The % Formazan production of positive control SDS is <=20% of negative control and the variation within replicates (RSD) is < 18%.

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

Duration of treatment / exposure:

60 minutes at 37 +/-1°C.

Duration of post-treatment incubation (if applicable):

18 hours post-incubation period at 37°+/-1°C, 5 +/-0.5% CO2

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

mean value

Value:

100.9

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:

MTT VIABILITY ASSAY RESULTS
- The test item is considered as not skin irritant.
- After the treatment, the relative absorbance values were increased to 100.9%. This value is well above the threshold for skin irritation (50%).The optical density of the negative control was well within the required acceptability criteri-on of 0.8 ≤ mean OD ≤ 2.8, OD was 1.8. The positive control induced a decrease in the relative absorbance as compared to the negative control to 3.3 % (required: ≤ 20%) for thus ensuring the validity of the test system. Variation within replicates was within the ac-cepted range for negative control, positive control and test item.

Any other information on results incl. tables

Table 7.3.1/&: Absorbance Values negative control, test item and positive control (OD at 570 nm):

Designation	Measurement	Negative Con-trol	Test susbtance	Positive Control
Tissue 1	1	1.925	1.718	0.101
	2	1.922	1.727	0.102
Tissue 2	1	1.830	1.900	0.098
	2	1.857	1.924	0.097
Tissue 3	1	1.804	1.977	0.087
	2	1.800	1.989	0.088

Table 7.3.1/2 Mean Absorbance Values

Designation	Negative Control	Test susbtance	Positive Control
Mean – blank (tissue 1)	1.888	1.687	0.066
Mean – blank (tissue 2)	1.808	1.876	0.062
Mean – blank (tissue 3)	1.766	1.947	0.052
Mean of the three tissues	1.821	1.837	0.060
Relative standard deviation of the three tissues	3.4%	7.3%	12.0%

Table 7.3.1/3 % Formazan Production

Designation	Test susbtance	Positive Control
% Formazan production (tissue 1)	92.6%	3.6%
% Formazan production (tissue 2)	103.0%	3.4%
% Formazan production (tissue 3)	106.9%	2.9%
% Formazan production (mean)	100.9%	3.3%

 

Table 7.3.1/4 Validity criteria and results

Criterion	Demanded	Found
OD of negative control	≥ 0.8 and ≤ 2.8	1.8
% Formazan production of positive control SDS	≤ 20% of negative control	3.3%
Variation within replicates (RSD)	< 18%	3.4% (negative control) 12.0% (positive control) 7.3% (test item)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, test material is not classified according to Regulation (EC) No. 1272/2008 (CLP).

Executive summary:

An in vitro skin irritation test using the Reconstructed Human Epidermis (Episkin Standard model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

 

The test item was applied at the dose of 30 µL, to 3 tissues of the Human skin model EpiDerm during 60 minutes, followed by a rinse with 25 mL of PBS and a 18 hours post-incubation period at 37+/-1°C, 5 +/-0.5% 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.

 

The mean corrected percent viability of the treated tissues was 100.9%, versus 3.3% in the positive control (5% Sodium Dodecyl Sulfate).

 

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin.