Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2019
Deviations:
no
GLP compliance:
no

Test material

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

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm (TM) reconstituted human epidermis
Justification for test system used:
according to OECD guideline (439)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- freeze-killed control tissues
- N. of replicates : 3 (for negative control and treatment group, each)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues treated concurrently with sterile PBS.
- The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and posttreatment incubation is less than or equal to 50%. Further testing with the Skin Corrosion Test (SCT) is required, because the SIT cannot resolve between UN GHS Categories 2 or 1.
- A single test composed of at least three tissue replicates should be sufficient for a test chemical when the result is unequivocal.
- 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 one in case of discordant results between the first two tests.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): Unchanged test substance

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% (w/v)
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 - hour post-incubation
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
compared to negative controls
Run / experiment:
tissue 1
Value:
102.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
compared to negative controls
Run / experiment:
tissue 2
Value:
92.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
compared to negative controls
Run / experiment:
tissue 3
Value:
97
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: Yes
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: 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:
Under the condition of the study, the test substance does not show a skin irritation potential.
Executive summary:

In the present study, the skin irritation potential of the test substance was investigated in accordance with TG OECD 439. Three human reconstituted epidermis tissues (EpiDerm™) were treated with 30 µL undiluted test substance for 1 hour followed by a 42-hour post-incubation period, followed by a cell viability (MTT) test. In addition to the test substance, control tissues were applied concurrently with 30 µL sterile PBS as negative control or with 30 µL 5% SDS as positive control to three tissues each. The optical density of the extracts of the tissues treated with the test substance was compared to negative control values from tissues treated with PBS, expressed as relative tissue viability. Based on the result of a pretest, the test substance is able to reduce MTT directly. Therefore, additional MTT reduction controls (freeze-killed control tissues) were applied concurrently with 30 µL sterile PBS or undiluted test substance (3 tissues each). However, the results of the killed control tissues did not indicate an increased MTT reduction.

Incubation of the reconstituted tissues with the test substance resulted in a mean viability of 97.3% (+/- 4.9% SD) compared to respective control tissue viabilities. Treatment with the positive control resulted in a mean viability of 2.1% (+/- 0.2% SD) compared to respective control tissue viabilities.

Based on the results observed and by applying the evaluation criteria described, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.

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