D,L-DIETHYL TARTRATE

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-14 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 439 without any deviation.

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

27 April 2017

Test material

Test material information

Constituent 1

Reference substance name:

D,L-DIETHYL TARTRATE

EC Number:

629-770-1

Cas Number:

57968-71-5

Molecular formula:

C8H14O6

IUPAC Name:

D,L-DIETHYL TARTRATE

Test material form:

liquid

Details on test material:

-Physical state: colourless - yellow liquid
-Storage condition of test material: Store in original containers protected from humidity. Store at ambient temperature (> 11°C).
- Density 20/4: 1,22
- Water solubility: 1410 g/L at 25°C (iSafeRat® Holistic HA-QSAR v1.5)

Specific details on test material used for the study:

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

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 met hod 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: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 17-RHE-094
- Production date: 12 September 2017
- Shipping date: 12 September 2017
- Delivery date: 12 September 2017
- Expiry date: 18 September 2017
- Date of initiation of testing: 12 September 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 42 minutes after the test item application, the nylon mesh was removed and the human epidermises were washed with 25 x 1 mL of DPBS. The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 2 hours and 55 minutes
- 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 reported
- Linear OD range of spectrophotometer: not reported.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.2 (> 0.7)
- Barrier function: ET50 = 5.1h (4.0h ≤ ET50 ≤ 10.0h )
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a stratum corneum
- Contamination: absence of mycoplasma
- Reproducibility: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: 3 living human skin models

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not a MTT reducer

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 42 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 42 minutes of exposure.

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
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL

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

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours post-incubation period at 37 °C, 5% CO2

Number of replicates:

Triplicate tissues for test substance, negative and positive controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

42 minutes exposure

Value:

65.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

2.4%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control OD of the 3 replicates is > 0.4 and < 1.5 (values between 0.738 and 1.008)
- Acceptance criteria met for positive control: yes, the positive control is classified as irritant
- Acceptance criteria met for variability between replicate measurements: yes, the SD values of the % viability are ≤ 18% (values between 0.9 and 17.3%)
The quality criteria required for acceptance of results in the test were satisfied.
The positive and negative control OD were within the historical control range.

Any other information on results incl. tables

Table 7.3.1/1: Assessment of the skin irritation - individual and average values of OD after 42 minutes exposure

 

Items	Skin	OD	Mean OD/disc#	Mean OD/product	Viability%	Mean viability%	SD	Conclusion
Negative control	1	0.939	1.008	0.841	119.8	100.0	17.3	-
		1.050
		1.035
	2	0.729	0.738		87.7
		0.776
		0.711
	3	0.819	0.778		92.5
		0.742
		0.775
Positive control	4	0.019	0.019	0.020	2.3	2.4	0.9	Irritant
		0.020
		0.019
	5	0.013	0.013		1.5
		0.014
		0.012
	6	0.041	0.028		3.3
		0.021
		0.023
Test item	7	0.787	0.621	0.553	73.8	65.8	8.7	Non irritant
		0.570
		0.507
	8	0.576	0.564		67.0
		0.568
		0.549
	9	0.454	0.475		56.5
		0.481
		0.492

 

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

OD: optical density

 

Acceptability criteria:

SD ≤ 18%

Negative control: OD value of the 3 replicates in the range ≥ 0.8 and ≤ 3.0.

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to OECD Guideline 439 and in compliance with GLP to evaluate the possible irritating effects of the test item after topical application on the in vitro human reconstructed epidermis SkinEthic RHE^® model.

 

Triplicate tissues were treated with 16 µl of the test item for an exposure period of 42 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. After MTT-loading, acidified isopropanol was then used for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period, the measurement of optical density of triplicate samples was measured at 570 nm.

 

The relative mean viability of the test item treated tissues was 65.8% after the 42‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The relative mean tissue viability for the positive control treated tissues was 2.4% relative to the negative control treated tissues and the standard deviation value of the viability was 0.9%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.841 and the standard deviation value of the viability was 17.3%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 8.7%. The test item acceptance criterion was therefore satisfied.

All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory. This was taken to show the correct functioning of the test system.

Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.