1,1-dimethoxycyclododecane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 - 22 Sep 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

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:

adopted 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

1,1-dimethoxycyclododecane

EC Number:

213-448-9

EC Name:

1,1-dimethoxycyclododecane

Cas Number:

950-33-4

Molecular formula:

C14H28O2

IUPAC Name:

1,1-dimethoxycyclododecane

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (Epi-200)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 19674 Kit B
- Delivery date: 17 Sep 2014
- Date of initiation of testing: 17 Sep 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile hood

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 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 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT was prepared on the day of testing using MTT-100 Assay Kit Components.
- Incubation time: 42 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 ± 1 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.5 h.
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is less than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

approximately 42 h

Number of replicates:

triplicates for each treatment and control group

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test substance was not considered to be a MTT reducer.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.742, 1.808 and 1.904) was in the range of ≥ 0.8 and ≤ 2.8 for 60 minutes treatment, thus showing the quality of tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 6.3% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The standard deviations of the % variabilities of the test substance, the positive and negative controls were < 10%, thus ensuring the validity of the study.

Any other information on results incl. tables

Table 2. Results after treatment

Dose Group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Rel. Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]***
Negative Control	60 min	1.742	1.808	1.904	1.818	95.8; 99.5; 104.7	4.5	100.0
Positive Control	60 min	0.126	0.117	0.103	0.115	6.9; 6.4; 5.7	9.7	6.3
Test Item	60 min	1.889	1.740	1.877	1.835	103.9; 95.7; 103.2	4.5	101.0

* Mean of three replicate wells after blank correction

** relative absorbance per tissue [rounded values]: (100 x (absorbance tissue)) / (mean absorbance negative control))

*** relative absorbance per treatment group [rounded values]: (100 x mean absorbance test item/positive control) / (mean absorbance negative control)

 

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. The mean relative absorbance value of the test item, corresponding to the cell viability, was not reduced (101.0%; threshold for irritancy: ≤ 50%), consequently, the test item was not irritant to skin.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

In the skin irritation study in vitro, the treatment with the test substance resulted in a cell viability of 101% and thus the test substance does not possess any skin irritating potential.