Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 December 2017-10 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name: Oxazolin (A 45 A)
Chemical name (name used in the study): 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol
Batch No.: UR010-076
Molecular Formula: C23H43NO2
Molecular Weight: 365.59 g/mol

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiSkinTMSM, EPISKIN SNC Lyon, France.
Details on animal used as source of test system:
Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV
collagen.
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
physiological saline
Details on test system:
Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37±1 °C for 42 hours in an incubator with 5±1 % CO2. The viability of each disk was assessed by incubating the tissues for 3 (±5 min) hours with MTT solution at 37±1 °C in 5±1 % CO2 and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.
SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control.
The test item has an intrinsic colour (brown), therefore two additional test item treated tissues were used for the non-specific OD evaluation.
The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed
tissues) were used to detect and correct for test substance interference with the viability measurement.
The test item is a possible MTT-reducer and has an intrinsic colour (brown). To avoid a possible double correction [TODTT (MTT and
NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Pre-incubation (day [-1]-day 0):
Assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed above the media in a separately prepared well.


Application (day 0):
- Test Item
A volume of 10 μL undiluted test item was applied evenly to the epidermal surface of each of the three test skin units.
- Positive and negative control
A volume of 10 μL positive control (SDS 5 % aq.) or negative control (1 x PBS) was applied to the skin surface by using a suitable pipette.
Duration of treatment / exposure:
Exposure (day 0): The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
Post-incubation (day 0-2):
After rinsing the units were placed into the plate wells filled with fresh pre-warmed “maintenance medium” (2 mL/well)
below them and then incubated for 42 hours at 37±1°C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere.
Number of replicates:
Number of replicate wells:
- 3 replicates per test item
- 3 replicates negative controls,
- 3 replicates positive controls,
- 2 replicates colour controls and 2 replicates non-specific colour control
- 3 killed treated tissues and 3 killed negative control tissues (for the MTT evaluation)

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item
Value:
> 77 - < 93
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Cell viability

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells are presented below:

OD values and viability percentages of the controls:

Substance

Optical Density (OD)

Viability (%)

Negative Control:1x PBS

1

1.12818

98

 

2

1.26353

109

 

3

1.07298

93

 

mean

1.15490

100

standard deviation (SD)

8.49

Positive Control:SDS (5 % aq.)

1

0.09848

9

 

2

0.06408

6

 

3

0.06213

5

 

mean

0.07490

6

 

standard deviation (SD)

1.77

OD values and viability percentages of the test item (including corrected values):

Test Item

Optical Density (OD)

TODTT

Viability (%)

Relative Viability (%)

4-ethyl-2- (8-heptadecenyl)-2- oxazoline-4- methanol

1

0.90010

0.89788

78

78

 

2

1.07165

1.06943

93

93

 

3

0.88670

0.88448

77

77

 

mean

0.952817

0.95060

83

82

 

standard deviation (SD)

 

8.93

8.93

OD values of additional controls for MTT-interacting test item:

Additional controls

Optical Density (OD)

Negative control killed tissues:1x PBS

1

0.10193

 

2

0.05473

 

3

0.05963

 

mean

0.07210

Test item treated killed tissues:4-ethyl-2-(8-heptadecenyl)-2- oxazoline-4-methanol

1

0.05955

 

2

0.10325

 

3

0.06015

 

mean

0.07432

OD values and NSC % of additional control:

Additional colour control

Optical Density (OD)

Non Specific Colour %(NSC %)

4-ethyl-2-(8-heptadecenyl)-2- oxazoline-4-methanol(test item treated tissues without MTT incubation)

1

0.00905

2.8

 

2

0.05575

 

mean

0.03240

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin
irritation potential under the utilized testing conditions. According to the current OECD Guideline No. 439, 4-ethyl-2-(8-
heptadecenyl)-2-oxazoline-4-methanol is considered as non-irritant to skin and is therefore not classified.
Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol on reconstituted human epidermis in the EPISKIN model in vitro.

Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37±1 °C for 42 hours in an incubator with 5±1 % CO2. The viability of each disk was assessed by incubating the tissues for 3 (±5 min) hours with MTT solution at 37±1 °C in 5±1 % CO2 and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control.

The test item has an intrinsic colour (brown), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item is a possible MTT-reducer and has an intrinsic colour (brown). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 82 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.