Santalene oil: fermentation products of glucose with santalene synthase modified Rhodobacter sphaeroides, distilled

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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)

Version / remarks:

2019

Deviations:

no

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Santalene oil: fermentation products of glucose with santalene synthase modified Rhodobacter sphaeroides, distilled

EC Number:

950-969-7

Molecular formula:

not applicable

IUPAC Name:

Santalene oil: fermentation products of glucose with santalene synthase modified Rhodobacter sphaeroides, distilled

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method (EIT) and considerations regarding applicability: according to the Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye Damage and Eye Irritation
-Test system: Three-dimensional human cornea model (EpiOcular (TM), i.e. a non-keratinized tissue construct composed of normal human-derived epidermal keratinocytes (surface 0.6 cm²).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: MTT reduction control (freeze-killed control tissues) applied with sterile deionized water or undiluted test substance

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL
- Concentration: undiluted test substance

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

2

Details on study design:

Details of the test procedure used:

-Two EpiOcular TM tissues were pretreated for 30 minutes with 20 µL PBS to wet the tissue surface. The tissues were incubated at standard culture conditions. Thereafter, the tissues were treated with 50 µL undiluted test substance, covering the whole tissue surface for 30 minutes followed by a 2-hour post-incubation period.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2 tissues each
- Due to the ability of the test substance to reduce MTT directly, freeze killed control tissues were treated with negative control and test substances (2 tissues each) in parallel.
- Induced cytotoxicity (= loss of viability) is measured by a colorimetric (MTT) assay, i.e. a reduction of mitochondrial dehydrogenase activity to reduce 3-[4, 5-d imethylthiazol-2-yl]-2 ,5-diphenyltetrazolium bromide (MTT) to the insoluble blue-colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically (wavelenght 570 nm). Optical density of the extracts of the tissues treated with the test substance is compared to values from negative control tissues and expressed as relative tissue viability.


Evaluation of results:
- The irritation potential of the test material is predicted from the mean relative tissue viabilities compared to the negative control tissues treated concurrently with sterile water.
- A chemical is considered as "non-irritant" (no UN GHS Category) if the mean relative tissue viability with a test material is greater than 60%.
- No prediction can be made, if the mean relative tissue viability with a test material is less or equal to 60%. Further testing with other test methods (e.g. BCOP) is required, because the EpiOcular EIT shows a
certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.
- In case of borderline results such as non-concordant replicate measurements and/or mean percent tissue viability equal to ± 5% of the cut-off value, a second test should be considered as well as a third test in case of discordant results between the first two tests. A "borderline" evaluation (60 ± 5%) was determined statistically using historical data of the performing laboratory and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 492.
- A single test composed of at least 2 tissue replicates is considered sufficient for a test chemical when the result is unequivocal.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent tissue viability 

Remarks:

compared to negative controls

Run / experiment:

viable tissue 1

Value:

98.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

percent tissue viability 

Remarks:

compared to negative controls

Run / experiment:

viable tissue 2

Value:

102.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct MTT reduction: The test substance is able to reduce MTT directly. Therefore, additional MTT reduction controls (freeze-killed control tissues) were applied concurrently with 50 µL sterile deionized water or with 50 µL undiluted test substance to 2 tissues each. The results of the freeze-killed control tissues indicate an increased MTT reduction (relative mean tissue viability 0.1 % of the negative control). Thus, for the test substance, the final mean viability is given after killed control correction.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

Historic control values of negative and positive controls were collected over an appropriate period. These data demonstrate the reproducibility of results and robustness of the procedures used.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

For details on results, please see table 1 in section "Attached background material".

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the present in vitro study, it was concluded that the test substance does not show an eye irritation potential.

Executive summary:

In the present study, the eye irritation potential of the test substance was investigated in accordance with TG OECD 492 (Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage).

Two EpiOcular™ tissues were treated with 50 µL undiluted test substance, covering the whole tissue surface for 30 minutes followed by a 2-hour post-incubation period, and a subsequent cell viability (MTT) test. In addition to the test substance, control tissues were applied concurrently with 50 µL sterile deionized water as negative control or with 50 µL methyl acetate as positive control to 2 tissues each. The optical density of the extracts of the tissues treated with the test substance was compared to negative control values, expressed as relative tissue viability. Based on the result of a pretest, the test substance is able to reduce MTT directly. Thus, two freeze-killed control tissues were treated additionally with each the test item and the negative control in the same way as the viable tissues. The results of the freeze-killed control tissues indicate an increased MTT reduction (relative mean tissue viability 0.1 % of the negative controls). Thus, the final mean viability values for test substance treated tissues were corrected accordingly.

Incubation of the reconstituted tissues with the test substance resulted in a mean viability of 100.6% (+/- 4.3% inter-tissue variability) compared to respective control tissue viabilities. Treatment with the positive control resulted in a mean viability of 20.7% (+/- 7.0% inter-tissue variability) compared to respective control tissue viabilities.

Based on the results observed in the EpiOcular™ test alone and by applying the evaluation criteria, it was concluded that the test substance does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.