Reaction mass of stigmast-5-en-3-β-ol and stigmasta-5,22-dien-3-β-ol and (24R)-ergost-5-en-3β-ol

Skin irritation / corrosion

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

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (24R)-ergost-5-en-3β-ol and stigmast-5-en-3-β-ol and stigmasta-5,22-dien-3-β-ol

EC Number:

904-790-6

Molecular formula:

C28H48O; C29H50O; C29H48O

IUPAC Name:

Reaction mass of (24R)-ergost-5-en-3β-ol and stigmast-5-en-3-β-ol and stigmasta-5,22-dien-3-β-ol

Specific details on test material used for the study:

Batch no. YH18ZC040304
Expiry date January 2021
Storage conditions Room temperature

In vitro test system

Test system:

human skin model

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Duration of treatment / exposure:

15 minutes

Observation period:

42 +/- 1 hour recovery period

Details on study design:

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of
the test item with the test system. In a first step, the test item was assayed for the ability of
reducing MTT per se. Light purple solution was noted in the MTT solution at the end of the
incubation period, indicating that the test item could direct interact with MTT. In a second
step, the test item was assayed for the ability of colouring water per se. A white opaque
suspension was observed. This suspension was not dispensable with automatic pipettes,
thus spectrophotometric analysis was not performed. Based on these results, additional
controls were added in the Main Assay.
In the Main Assay, the test item was applied as supplied in three replicates at the treatment
level of 20±2mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 µL/cm2).
Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and
Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in
the same number of replicates and test conditions at the treatment level of 20 µL/epidermis
unit. In order to verify if the test item results had to be corrected, the non specific colour
(NSCliving) was evaluated using two alive treated tissues without MTT staining and compared
with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated
using two killed tissues and compared with negative control performed with alive tissues.
Since the test item is able both to stain tissue and reduce MTT, to avoid a possible double
correction for colour interference, a third control for Non Specific Colour in killed tissue
(NSCkilled) was performed.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main

Value:

100

Other effects / acceptance of results:

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of
the test item with the test system. In a first step, the test item was assayed for the ability
of reducing MTT per se. A white precipitate was noticed, however no colour change was
observed in the MTT solution at the end of the incubation period, indicating that the test
item could not direct interact with MTT. In a second step, the test item was assayed for the
ability of colouring water per se. A white opaque suspension, was obtained. This suspension
was not dispensable with automatic pipettes, thus spectrophotometric analysis was not
performed. Based on this result, additional controls for colour interference were added in
the Main Assay.
In the Main Assay, the test item was applied as supplied in three replicates at the treatment
level of 20 ± 2 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2).
Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and
Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in
the same number of replicates and test conditions at the treatment level of 20 µL/epidermis
unit. In order to verify if the test item results had to be corrected, the non specific colour
(NSCliving) was evaluated using two alive treated tissues without MTT staining and compared
with the D-PBS control.

Any other information on results incl. tables

In the Main Assay, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According
to the method, the negative control mean value is considered the baseline value of the
experiment and thus represents 100% of cell viability.
The positive control caused the expected cell death (7% of cell viability when compared to the negative control) and variability (SD of % viability equal to 2.05). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤ 18), the assay was regarded as valid.

The NSCliving value was 4%, thus only the OD-blank background subtraction was performed.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not induce cell death in any replicate, the mean cell viability after the blank
subtraction was 100%, when compared to the negative control. Intra-replicate variability
was acceptable with a SD of % viability value equal to 1.88 (lower than 18, as stated in the
Study Protocol).
Based on the results obtained, the test item Mixture of phytosterols is classified as non-
irritant to the skin (UN GHS No Category).