Dimethyl azelate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2017 - 20 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 439 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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 28 July 2015

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:

23 July 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

French GLP Compliance Programme for chemical products (inspected on 30-31 January 2017 / signed on 27 April 2017)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl azelate

EC Number:

217-060-0

EC Name:

Dimethyl azelate

Cas Number:

1732-10-1

Molecular formula:

C11H20O4

IUPAC Name:

1,9-dimethyl nonanedioate

Test material form:

liquid

Details on test material:

Name: Dimethyl Azelate
Batch no.: 1602100006
Appearance: Oily liquid, colorless
CAS No.: 1732-10-1
EINECS-No.: 217-060-0
Molecular formula: C11H20O4
Molecular weight not stated
Purity: 86 %w/w
Production date: 04 October 2016
Expire date: 1 November 2018
Storage: Fridge (2-8°C), keep away from humidity

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 17-RHE-088
- Production date: Not reported
- Shipping date: Not specified
- Delivery date: 29 August 2017
- Expiry date: 04 September 2017
- Date of initiation of testing: 23 August 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 x 1 mL of DPBS (Dutscher - Batch No. 9130617)
- Observable damage in the tissue due to washing: The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues.
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek
- Wavelength: 570 nm
- Filter: Not applicable
- Filter bandwidth: Not applicable
- Linear OD range of spectrophotometer: No data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD570= 1.4, CV= 4.3% (specification: OD > 0.7)
- Barrier function: ET50 (Exposure time inducing 50% viability using Triton X-100 1%)= 4.3h (specification: 4.0h =< ET50 =< 10.0h)
- Morphology: well differenciated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: : absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- There is no direct interaction between the test item and MTT.
- No need to add non-specific coloration control since the test item has no coloration potential.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes of exposure and 42 hours of post-treatment incubation is > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL of the test item was then applied to the epidermal surface.
- Concentration (if solution): not applicable

VEHICLE
not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL of DPBS
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL of SDS
- Concentration (if solution): 5% w/v aqueous solution

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 42 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 41 hours in fresh medium.

Number of replicates:

3 living tissues for test item, negative and positive controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean corrected percent viability

Run / experiment:

1

Value:

108.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

1.2%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not specified
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

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: Provided in the study report

Any other information on results incl. tables

Table 7.3.1/1: Mean OD₅₇₀Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD	Conclusion
Negative control	1	0.750 0.851 0.872	0.824	0.787	104.7	100.0	5.2
	2	0.770 0.795 0.813	0.793		100.8
	3	0.759 0.745 0.726	0.743		94.4
Positive control	4	0.008 0.009 0.010	0.009	0.009	1.1	1.2	0.2	Irritant
	5	0.008 0.009 0.008	0.008		1.0
	6	0.010 0.010 0.013	0.011		1.4
Test item	22	0.764 0.827 0.834	0.808	0.854	102.7	108.6	6.6	Non irritant
	23	0.927 0.896 0.907	0.910		115.7
	24	0.853 0.855 0.823	0.844		107.3

#: mean of 3 values (triplicate of the same extract)

OD: optical density

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item Dimethyl Azelate has to be considered as non-irritant to skin according to CLP regulation (EC) No.1272/2008. It is also not classified according to UN GHS regulation.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439, the EU Method B.46 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model.

The test item Dimethyl Azelate was applied, as supplied, at the dose of 16 μL to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 41 hours post-incubation period at 37°C, 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 percent viability of the treated tissues was 108.6%, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate).

 

Under the experimental conditions of this study, the test item Dimethyl Azelate has to be considered as Non-irritant to skin to skin according to CLP regulation (EC) No.1272/2008. It is also not classified according to UN GHS regulation.