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Toxicological information

Eye irritation

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Administrative data

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

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 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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
CAS No. 68140-98-7
EINECS-No. 268-820-3
Molecular formula C23H43NO2
Molecular weight 365.59 g/mol
Vapour pressure < 0.001 hPa, 20 °C
Stability Stable under normal conditions
Solubility < 0.05 g/L, not soluble
Production date 01. Sep. 2017
Batch no. # 170903
Composition 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol
Purity 80.6 %
Homogeneity homogeneous
Expiry date 31. Aug. 2019

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results
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)

Applicant's summary and conclusion

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.