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:

From June 22 to 26, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD test Guideline No. 439 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other 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)

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on October 13 and 14, 2014 / signed on April 08, 2015

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl tartrate

EC Number:

201-783-3

EC Name:

Diethyl tartrate

Cas Number:

87-91-2

Molecular formula:

C8H14O6

IUPAC Name:

diethyl tartrate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Tartrate diethyle
- Physical state: Incolor liquid
- Storage condition of test material: At room temperature (20 ± 5 °C) in the dark; test item was stored in the test facility in a closed vessel at room temperature (18.9-25.8 °C) in the dark.

Test animals

Species:

other: human skin model EPIDerm™ tissues

Details on test animals or test system and environmental conditions:

Commercially available EPIDerm™-Kit (EPI-200-SIT): The EPIDerm™ tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EPIDerm™ tissues are cultured on specially prepared cell cultures inserts.
Origin: EPIDerm™ tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Day of delivery: 23 June 2015
Batch no.: 21677

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

other: not applicable

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of the test item was applied to the epidermis surface
- Concentration: The test item was used as supplied.

Number of animals:

Triplicate tissues for test item, negative and positive controls

Details on study design:

TEST VESSELS
All vessels used are made of single use sterile plastic.
The following vessels were used: 6-well-plates; 24-well plates; 96-well-plate

PERFORMANCE OF THE STUDY
Pre-Incubation of Tissues
Eight 6-well-plates were prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Viable tissues were transferred (3 per plate) in the wells with the medium using sterile forceps under the clean bench and placed into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 1 h. After the pre-incubation (1 h), the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1 °C and 5 ± 0.5% CO2 for 18 h.

Treatment
The pre-incubated tissues were placed into fresh 6-well-plates containing 0.9 mL assay medium per well, using the upper row only.
One 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 transferred into the incubator for 35 min. at 37 ± 1 °C and 5 ± 0.5% CO2. 60 min. after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately 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 at 37 ± 1 °C and 5 ± 0.5% CO2 for 24 h.

Medium Renewal
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 incubator for 18 h and 10 min. for post-incubation at 37 ± 1 °C and 5 ± 0.5% CO2.

MTT Assay
After a total incubation time of 42 h, a 24-well-plate was prepared with 300 μL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator at 37 ± 1 °C and 5 ± 0.5% CO2 for 3 h. After this time, the MTT reagent was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times.
At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 h at room temperature.
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-wellplate which was read in a plate spectral photometer at 570 nm.

CONTROLS (reference substances)
- Negative: One 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 surface.
- Positive: One 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.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

102.9

Other effects / acceptance of results:

Skin Irritation Potential of the Test Item: The mean tissue viability as percentage of mean OD of the negative control was 102.9% (± 3.2% SD) after the treatment with the test item. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as not irritant to skin.

In vivo

Other effects:

None

Any other information on results incl. tables

Table 7.3.1/1: Absorbance Values negative control, test item and positive control (OD at 570 nm)

Designation	Measurement	Negative Control	Test item	Positive Control
Tissue 1	1	2.058	2.029	0.122
	2	2.031	2.045	0.121
Tissue 2	1	2.021	2.061	0.107
	2	2.001	2.095	0.107
Tissue 3	1	1.844	1.952	0.119
	2	1.851	1.964	0.124

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol (0.036). Mean and relative standard deviation (comparison of the three tissues) was also calculated.

 

Table 7.3.1/2: Mean Absorbance Values

Designation	Negative Control	Test item	Positive Control
Mean corrected (tissue 1)	2.009	2.001	0.086
Mean corrected (tissue 2)	1.975	2.042	0.071
Mean corrected (tissue 3)	1.812	1.922	0.086
Corrected mean of the three tissues	1.932	1.988	0.081

 

Assessment of viability

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

 

Table 7.3.1/3: % Formazan Production

Designation	Negative Control	Test item	Positive Control
% Formazan production (tissue 1)	104.0%	103.6%	4.5%
% Formazan production (tissue 2)	102.2%	105.7%	3.7%
% Formazan production (tissue 3)	93.8%	99.5%	4.5%
% Formazan production (mean)	100%	102.9%	4.2%
± SD of mean Formazan production (%)	5.5%	3.2%	0.5%

 

The mean percentage values of formazan production give the mean viability of the tissues.

Validity and Acceptability

 

Table 7.3.1/4: Validity

Criterion	Demanded	Found
OD of negative control	≥ 0.8 and ≤ 2.8	1.9
% Formazan production of positive control SDS	≤ 20% of negative control	4.2%
SD of mean viability of the tissues replicates (%)	< 18%	5.5 % (negative control) 0.5 % (positive control) 3.2 % (test item)

 

All validity criteria were met.

Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, test item was not classified as irritant according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the human skin model EPIDerm™. 

 

Three tissues of the human skin model EPIDerm™ were treated for 60 min with 30 μL of the undiluted liquid test item (using a nylon mesh) applied to each tissue and spread to match the tissue size (0.63 cm²). DPBS-buffer was used as negative control and 5 % SDS solution was used as positive control. After rinsing of the test item the tissues were incubated for 42 hours. The cell viability was determined by dehydrogenase conversion of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) to a blue formazan product. The percentage viability values, compared to negative control, were used to identify irritant and non-irritant substances.

 

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.9. The positive control showed clear irritating effects. Relative absorbance was reduced to 4.2 %. Variation within tissues was acceptable (< 18%). Therefore this assay was valid since negative and positive controls showed results within the acceptable range.

 

After the treatment with test item, the mean tissue viability was 102.9 % (± 3.2 % standard deviation). This value is well above the threshold for irritation potential (50%). Therefore, test item is considered as not skin irritant in the Human Skin Model Test.

 

Under the test conditions, test item was classified as irritant according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).

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