Dimethyl azelate

Endpoint summary

Description of key information

Skin irritation: not irritant to skin (OECD 439, GLP, K, rel.1)

Eye irritation/damage: no classification required for eye irritation or serious eye damage (OECD 492, GLP, K, rel. 1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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.

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

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 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

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Sepember 2017 - 08 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 492 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 28 July 2015

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)

Species:

other: Reconstructed human cornea-like epithelium tissues (EpiOcular™ tissue model)

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. This test guideline is applicable to liquids and semi-solids, so is considered to be applicable to the test item.

CELL SYSTEM:
- EpiOcular™ OCL-212-ver2.0, supplied by MatTek Corporation (Bratislava, Slovakia).
- Lot No.: 27006
- Keratinocyte strain: 4F1188
- Analysis for potential biological contaminants: none detected (HIV-1, Hep. B and Hep. C virus; Bacteria, yeast and other fungi)
- Tissue viability: 1.534, within the acceptance criteria (1.1-3.0)
- Barrier function: 17.77 min, within the acceptance criteria (12.2-37.5)
- Sterility: Sterile
- Transport: Not specified
- Storage: Not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): not applicable

VEHICLE: Not applicable

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

- 12-minute immersion period at room temperature
- 2 hour and 12 minutes post-exposure incubation at 37°C, 5% CO2

Number of animals or in vitro replicates:

2 replicates

Details on study design:

- Details of the test procedure used : procedure for liquids
* Pre-treatment: after an overnight incubation, tissues were pre-wetted with 20 µL of Ca2+Mg2+Free-DPBS. Then tissues were incubated for 30 minutes at standard culture conditions.
* Treatment: 50 µL of test item, positive or negative control was applied to the entire surface of 2 living RhCE tissue replicates during 30 minutes at standard culture conditions.
* Post-exposure incubation period: after treatment, tissues were carefully washed by extensive rinsing with Ca2+Mg2+Free-DPBS. Rinsed tissues were checked for any coloration and for comparable colour with negative control treated tissues (whitish). The rinsing step was followed by a 12-minute immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. Then the RhCE constructs were incubated for 2 hours and 12 minutes post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

- Doses of test chemical and control substances used : 50 µL

- Duration and temperature of pre-treatment (30 minutes), exposure (30 minutes), post-exposure immersion (12 minutes) and post-exposure incubation (2 hour and 12 minutes)

- Description of any modifications to the test procedure : None

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : Since the test item did not interact with MTT, none killed control tissue models were added to the study. The test item did not show any coloration potential in presence of water or isopropanol, thus no non-specific coloration control was added to the study.

- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is identified potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).
According to OECD guideline 492 a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : Yes, attached to the study report.
* Historical negative control ranges 83.17 - 116.83%
* Historical positive control ranges 6.50 - 45.11%

- Complete supporting information for the specific RhCE tissue construct used : Yes, attached to the study report.

- Reference to historical data of the RhCE tissue construct : No

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : Yes, attached to the study report.

- Positive and negative control means and acceptance ranges based on historical data : Yes

- Acceptable variability between tissue replicates for positive and negative controls: Yes

- Acceptable variability between tissue replicates for the test chemical: Yes

Irritation parameter:

other: % Tissue viability

Remarks:

mean corrected percent viability

Run / experiment:

1

Value:

92.81

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.00%

Positive controls validity:

valid

Remarks:

43.31%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, OD values included between 0.848 and 1.169
- Acceptance criteria met for positive control: % viability= 43.31%
- Range of historical values if different from the ones specified in the test guideline:
- Negative control: OD values [0.645-1.563]
- Positive control: % viability [6.50-45.11]

Table 7.3.2/1: Mean OD₅₇₀Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

	Tissue	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Difference of viability %	Conclusion
Negative control	1	0.849 0.848 0.856	0.851	1.002	84.93	100.00	30.14
	2	1.169 1.135 1.157	1.153		115.07
Positive control	3	0.405 0.399 0.399	0.401	0.434	40.02	43.31	6.59	UN GHS Category 2 or 1
	4	0.467 0.463 0.471	0.467		46.61
Test item	5	0.970 0.902 0.914	0.932	0.930	93.01	92.81	0.40	No Category
	6	0.929 0.928 0.925	0.928		92.61

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

 

 

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

OD: optical density

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions adopted and in accordance with Regulation EC No. 1272/2008, test item, Dimethyl Azelate, does not require classification for eye irritation or serious eye damage according to CLP and UN-GHS regulations.

Executive summary:

A study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The study was conducted according to the OECD guideline No. 492 and in compliance with GLP.

The test item, Dimethyl Azelate, was applied as supplied at the dose of 50 µL to 2 living DPBS pre-treated RhCE (EpiOcular^TM tissue model) during 30 minutes at 37°C, 5% CO₂, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours and 12 minutes post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

The mean percent tissue viability of the RhCE replicates treated with the test item Dimethyl Azelate was 92.81 %, versus 43.31% in the positive control (Methyl acetate).

Under the experimental conditions adopted and in accordance with Regulation EC No. 1272/2008, test item Dimethyl Azelate does not require classification for eye irritation or serious eye damage according to CLP and UN-GHS regulations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

One key study was identified for the skin irritation. A key study (Floriot, 2017, Rel.1) was performed to evaluate the skin irritation potential of the test substance, Dimethyl Azelate. This in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the SkinEthic 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.

Eye irritation:

A key study was identified for eye irritation. This in vitro eye irritation test (Floriot, 2017, Rel.1) was performed according to the OECD Guideline 492 and in compliance with GLP, using the Reconstructed human Cornea-like Epithelium (RhCE).

The test item, Dimethyl Azelate,was applied as supplied at the dose of 50 µL to 2 living DPBS pre-treated RhCE (EpiOcular^TMtissue model) during 30 minutes at 37°C, 5% CO₂, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours and 12 minutes post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. The mean percent tissue viability of the RhCE replicates treated with the test item Dimethyl Azelatewas 92.81 %, versus 43.31% in the positive control (Methyl acetate).

Under the experimental conditions adopted and in accordance with Regulation EC No. 1272/2008, test item Dimethyl Azelate does not require classification for eye irritation or serious eye damage according to CLP and UN-GHS regulations.

Respiratory irritation:

No data was available regarding respiratory irritation assessment.

Justification for classification or non-classification

Harmonized classification:

Dimethyl Azelate does not have harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, the substance should not be classified as skin irritant according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the UN-GHS.

Based on the available information, the substance should not be classified as eye irritant according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the UN-GHS.

No data was available regarding respiratory irritation. However, the substance not being skin or eye irritant, no classification is expected for respiratory irritation.