C10-13-alkyl(branched, unsaturated)-succinic acid-4-(3-methyl-butyl)-ester

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-02-16 to 2016-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline and GLP conform well documented scientific study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28 July 2015

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

921-522-3

EC Number:

921-522-3

IUPAC Name:

921-522-3

Constituent 2

Reference substance name:

Tetrapropylene succinic acid monoisobutylester

IUPAC Name:

Tetrapropylene succinic acid monoisobutylester

Test material form:

other: viscous liquid

Details on test material:

Identification: Tetrapropylene succinic acid monoisobutylester
Appearance: Light brown, viscous liquid
Batch: ESD0018639
Purity/Composition: 93.0% (w/w)
Test item storage: At room temperature
expiry date: Stable under storage conditions until 30 November 2016

Specific details on test material used for the study:

Identification Tetrapropylene succinic acid monoisobutylester
Appearance Light brown, viscous liquid
Batch ESD0018639
Purity/Composition 93.0% (w/w)
Test item storage At room temperature
Stable under storage conditions until 30 November 2016 (expiry date)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2).
- Tissue batch number: Lot no.: 16-EKIN-007
- Production date: 2016-02-16
- Shipping date: n.a.
- Delivery date: n.a.
- Date of initiation of testing: 2016-02-16

TEMPERATURE USED FOR TEST SYSTEM
- On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 23 hours at 37°C
- 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: n.a.
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL in PBS; final concentration 0.3 mg/mL
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm


NUMBER OF REPLICATE TISSUES:
- Test was performed on a total of 3 tissues per test item together with negative and positive controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Tetrapropylene succinic acid monoisobutylester was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model and (project 511530). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Tetrapropylene succinic acid monoisobutylester did not interfere with the MTT endpoint

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

PREDICTION MODEL / DECISION CRITERIA
Relative mean viability of 3 individual tissues after 15 minutes of exposure and 42 hours of post incubation:
- The test substance is considered to be irritiant to skin Category 1 or Category 2 (additional information on corrosion needed) if the test item has ≤ 50% of the mean viability of the negative controls
- The test substance is considered to be non-irritatnt to skin (no category) if the test item has > 50% of the mean viability of the negative controls

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL: physiological saline
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 0.9%

POSITIVE CONTROL: SDS
- Amount(s) applied (volume or weight): 25µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature
The positive control was re-spread after 7 minutes contact time.

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

3

Test animals

Species:

human

Strain:

other: not relevant

Details on test animals or test system and environmental conditions:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 16-EKIN-007
Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

4.9

Vehicle controls validity:

other: no vehicle used, test item used undiluted

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Acceptability of the assay
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Data evaluation and statistical procedures
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Any other information on results incl. tables

Table 1. Data interpretation of test items

Relative mean viability of 3 individual tissues after 15 minutes of exposure and 42 hours of post incubation	Prediction to be considered
≤50% of the mean viability of the negative controls	Category 1 or Category 2 (additional information on corrosion needed)
> 50% of the mean viability of the negative controls	No category

 

Table 2. Mean absoption in the in vitro skin irritation test with Tetrapropylene succinic acid monoisobutylester

	Mean (OD570)
Negative control	0.892 ± 0.109
Tetrapropylene succinic acid monoisobutylester	0.043 ± 0.006
Positive control	0.086 + 0.045

Table 3. Mean tissue viability in the in vitro skin irritation test with Tetrapropylene succinic acid monoisobutylester

	Mean tissue viability (percentage of control)
Negative control	100
Tetrapropylene succinic acid monoisobutylester	4.9
Positive control	10

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It is concluded that Tetrapropylene succinic acid monoisobutylester is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In order to assess the possible skin irritation potential, Tetrapropylene succinic acid monoisobutylester was tested in vitro through topical application for 15 minutes on a human three dimensional epidermal in vitro skin model (EPISKIN Standard model). The study procedures described in this report were based on the most recent OECD 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 28 July 2015)). The test item was applied undiluted directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Skin irritation is expressed as the remaining cell viability after exposure to the test item.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 4.9%. Since the mean relative tissue viability for Tetrapropylene succinic acid was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant.The positive control had a mean cell viability of 10% after 15 minutes exposure.