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

Endpoint summary

Description of key information

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.

The test item 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4- methanol showed no effects on the cornea of the bovine eye.

Under the conditions of the EpiOcularTM Eye Irritation Test, 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol is considered non- eye irritant and is therefore not classified.

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:

13 December 2017-10 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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).

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:

10 μL undiluted test item


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)

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

OEDC 437

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2018-November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container within 1 hour.

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): without dilution

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10), batch no.: 20180607

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): Dimethylformamide, DMF, CAS-No. 68-12-2, undiluted, batch no.: 475235719

Duration of treatment / exposure:

Exposure time of test item on corneas: 10 minutes at 32 ± 1 °C.

Number of animals or in vitro replicates:

three replicates

Details on study design:

Closed Chamber Method
The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μL of the appropriate liquid through the refill hole in the holder on the cornea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item.
Exposure time of the test item on the corneas was 10 minutes at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation).
After post-incubation time, the cMEM without phenol red was renewed in both chambers. Then, the final opacity value of each cornea was recorded.

Permeability Test
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas.
For the closed chamber method, a sodium fluorescein solution with a concentration of 4 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C.
After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

SELECTION AND PREPARATION OF CORNEAS
- only corneas which were free from damages were used
- corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside.

NUMBER OF REPLICATES
- there replicates

NEGATIVE CONTROL USED
yes

POSITIVE CONTROL USED
yes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 2 hours at 32 ± 1 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD at 492nm)


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

VALIDITY: The test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean. The mean IVIS of the negative control has to show an IVIS ≤ 3.

Irritation parameter:

in vitro irritation score

Value:

0.39

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes: Hank’s Balanced Salt Solution (HBSS): no irritating effect on the cornea, IVIS (In Vitro Irritancy Score) = 1.34.

- Acceptance criteria met for positive control: yes: Dimethylformamide (DMF) undiluted: serious eye damage on the cornea, IVIS = 120.36 (within two standard deviations of the current historical mean)

Interpretation of results:

GHS criteria not met

Conclusions:

The test item 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol showed no effects on the cornea of the bovine eye. The calculated IVIS (In Vitro Irritancy Score) is 0.39. According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Executive summary:

The test item 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol was brought onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. Bovine corneas used in this study were collected from slaughtered cattle that were between 12 and 60 months old.

The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured. One valid experiment was performed.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) is 1.34.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 120.36.

Under the conditions of this study, the test item 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4- methanol showed no effects on the cornea of the bovine eye. The calculated IVIS is 0.39.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14. Jun.2018-15. Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

Pre-Tests
- Assessment of Direct Reduction of MTT by the Test Item: 3 hours
- Assessment of Coloured or Staining Test Items: 3 hours

Main Test: 28 minutes

Duration of post- treatment incubation (in vitro):

tissues were incubated for 115 minutes (main test)

Number of animals or in vitro replicates:

two tissue replicates

Irritation parameter:

other: relative tissue viability

Value:

110.3

Negative controls validity:

valid

Positive controls validity:

valid

In the pre-test, potential MTT reduction by the test item was observed. This is why an additional test was performed to exclude wrong photometrical measurement values due to direct MTT reduction by the test item.

The additional test showed that the MTT reduction by the test item did not influence the result of the main test.

Interpretation of results:

GHS criteria not met

Conclusions:

After treatment with the test item, the mean value of relative tissue viability was increased to 110.3%. This value is well above the threshold for eye irritation potential (≤ 60%).
Under the conditions of the test, 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

Executive summary:

The test item 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol was applied to a threedimensional human cornea tissue model in duplicate for an exposure time of 28 minutes.

50 μL of the liquid test item were applied to two tissue replicates. One valid experiment was performed.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

Demineralised water was used as negative control and methyl acetate was used as positive control.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.9. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 34.7 % (< 50%).

Variation within tissue replicates of the controls and the test item was acceptable (< 20%).

After treatment with the test item, the mean value of relative tissue viability was 110.3 %.

This value is well above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.

Under the conditions of the test, 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol is considered non- eye irritant in the EpiOcularTM Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)