[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20 June, 2017 to 6 September, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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 material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

450-320-3

EC Name:

-

Molecular formula:

C15H28O

IUPAC Name:

(3S,6E)- 3,7,11-trimethyldodeca-6,10-dien-1-ol

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™; reconstructed three-dimensional human epidermis (EPI-200)

Source species:

other:

Cell type:

other:

Cell source:

other:

Details on animal used as source of test system:

MatTek Corp. EpiDerm™ reconstituted human epidermis
The EpiDerm™ tissues were stored at 1-10ºC until used. On the day of receipt, an appropriate volume of fresh EpiDerm™ Assay Medium was pre-warmed to room temperature. Nine-tenths (0.9) mL of Assay Medium were aliquoted into the appropriate wells of each 6-well plate. Each EpiDerm™ tissue was inspected for air bubbles between the agarose gel and insert prior to opening the sealed package. The 24-well shipping containers were removed from the plastic bag and their surfaces were disinfected with 70% ethanol. An appropriate number of tissues were removed from the 24-well shipping plate and transferred to the 6-well plate containing Assay Medium. The plates were placed into the incubator at 37.0±0.5ºC in a humidified atmosphere of 5.0±0.2% CO2 in air (standard culture conditions) for 60±5 minutes. At the end of the incubation period, the inserts were transferred into wells containing fresh Assay Medium and incubated overnight (18 ±3 hours) to acclimate the tissues.

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL
- Concentration: undiluted test substance

Duration of treatment / exposure:

60 ± 1 minutes (25 minutes at room temperature/35 minutes at 37°C)

Duration of post-treatment incubation (if applicable):

42 ± 4 hours

Number of replicates:

3 tissues were used per treatment for the test substance, the negative control (NC) and the positive control (PC)

Test animals

Species:

other: not applivable; in vitro test

Strain:

other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200)

Details on test animals or test system and environmental conditions:

MatTek Corp. EpiDerm™ reconstituted human epidermis
The EpiDerm™ tissues were stored at 1-10ºC until used. On the day of receipt, an appropriate volume of fresh EpiDerm™ Assay Medium was pre-warmed to room temperature. Nine-tenths (0.9) mL of Assay Medium were aliquoted into the appropriate wells of each 6-well plate. Each EpiDerm™ tissue was inspected for air bubbles between the agarose gel and insert prior to opening the sealed package. The 24-well shipping containers were removed from the plastic bag and their surfaces were disinfected with 70% ethanol. An appropriate number of tissues were removed from the 24-well shipping plate and transferred to the 6-well plate containing Assay Medium. The plates were placed into the incubator at 37.0±0.5ºC in a humidified atmosphere of 5.0±0.2% CO2 in air (standard culture conditions) for 60±5 minutes. At the end of the incubation period, the inserts were transferred into wells containing fresh Assay Medium and incubated overnight (18 ±3 hours) to acclimate the tissues.

Test system

Type of coverage:

other: a nylon mesh was placed on each tissue surface to spread the liquid substances.

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL
- Concentration: undiluted test substance

Duration of treatment / exposure:

60 ± 1 minutes (25 minutes at room temperature/35 minutes at 37°C)

Observation period:

42 ± 4 hours

Number of animals:

3 tissues were used per treatment for the test substance, the negative control (NC) and the positive control (PC)

Details on study design:

The test substance was added to a 1.0 mg/mL MTT (DOJINDO Laboratories) solution in warm MTT Addition Medium to assess its ability to directly reduce MTT. Thirty microliters of the test substance were added to 1 mL of the MTT solution, and the mixture was incubated in the dark at 37ºC for 60 minutes. If the MTT solution color turned blue/purple, the test substance was presumed to have reduced the MTT. The test substance, L-Dihydrofarnesol, was not observed to directly reduce MTT in the absence of viable cells.
Prior to performing the assay, the compatibility of the test substance with the nylon mesh was evaluated. Nylon meshes (MatTek Corp.) were placed on a glass slide and 30 μL of the test substance was applied. Using a microscope, each mesh was checked after 60 minutes of exposure to assess any interaction between the test substance and the mesh.
The test substance, L-Dihydrofarnesol, was not observed to interact with the nylon mesh, and therefore a nylon mesh was used to aid in the spreading of the test substance after dosing the EpiDerm™ tissues.
Definitive Test - After the overnight incubation for 18±3 hours, the 6-well plates containing the EpiDerm™ tissues were removed from the incubator and placed into a biological safety cabinet at room temperature. The EpiDerm™ tissues were treated in triplicate with the test substance, L-Dihydrofarnesol, for 60±1 minutes. Thirty microliters of the test substance were applied to each of three tissues at 1 minute intervals per tissue. Immediately after the test substance administration onto the tissue, a nylon mesh was placed on each tissue surface to spread the substances. The EpiDerm™ tissues were tested in triplicate with the positive or negative control for 60±1 minutes. Thirty microliters of each control were applied to each of three tissues at 1 minute intervals per tissue. Immediately after control administration onto the tissue, a nylon mesh was placed on each tissue surface to spread the negative and positive controls. The plates with dosed tissues were kept in the biological safety cabinet until the last tissue was dosed. After the last tissue was dosed, all of plates were transferred to the incubator for 35±1 minutes at standard culture conditions. After 35 minutes, all of the plates were removed from the incubator, placed into the biological safety cabinet and kept at room temperature until the exposure period was completed for the first dosed tissue.
After 60 ± 1 minutes exposure, the EpiDerm™ tissues were rinsed with sterile, PBS(−) by filling and emptying the tissue insert 15 times. A stream of PBS(−) was directed onto the tissue surface. After the 15th rinse, each of the 3 inserts per treatment group (test substance, positive and negative control) was completely submerged, gently swirled, and rinse media dumped in a beaker containing approximately 150 mL of PBS(−) and specifically assigned for each treatment group; this procedure was repeated three times for each insert of each treatment group. Finally, the tissues were rinsed once more on the inside and outside of the tissue insert with sterile PBS(−). Remaining PBS(−) was removed from inside and outside of tissue insert with a sterile cotton swab. Tissue inserts were transferred to new 6-well plates containing 0.9 mL of fresh Assay Medium. PBS(−) on the surface of tissues were removed with a sterile cotton swab. The tissues were then placed into the incubator at standard culture conditions for a post-treatment expression incubation of 24±2 hours.
After the 24±2 hours post-treatment expression incubation, the 6-well plates were removed from the incubator. The tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh Assay Medium warmed to approximately 37ºC. The tissues were placed back into the incubator for an additional 18±2 hours post-treatment incubation at standard culture conditions.
MTT Test - A 1.0 mg/mL solution of MTT prepared in warm MTT Addition Medium was prepared just before use. Three hundred microliters of MTT solution were added to each well of a 24-well plate. At the end of the 42±4 hour incubation period, the EpiDerm™ tissues were transferred to the appropriate well containing 0.3 mL of MTT solution. The plates were incubated at 37±1ºC for 180 ± 5 minutes at standard culture conditions. After the MTT incubation, medium was removed from all wells. PBS(−) was added to the outsides of tissue insert and removed. This process was performed three times. All tissues were transferred into a new 24-well plate. Two milliliters of 2-propanol was added to each well. The plates were put into a plastic bag, and shaken for 2 hours at room temperature to extract the MTT. At the end of the extraction period, the extracts were moved from the inside of tissue inserts to plate and mixed to obtain homogeneous solutions. Two hundred microliters per well of the extracts were transferred into a 96-well plate (n = 2). Two hundred microliters per well of 2-propanol was used as blank (n = 6). The optical density at 570 nm (OD570) of each well was measured spectrophotometrically using Multimode Microplate Reader (FLUOstar OPTIMA, BMG LABTECH).

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

41.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Basis: mean. Time point: 60 minutes and a 42 hour post exposure incubation. Reversibility: no data. (migrated information)

Other effects / acceptance of results:

In vivo
The skin irritation potential of the test substance, L-Dihydrofarnesol, was evaluated by measuring the relative cell viability in treated EpiDerm™ tissues after a 60-minute exposure and a 42-hour post-exposure incubation.
The mean OD570 of the negative control, PBS(−), was 1.787. The mean viability of the positive control, 5% SDS, was 1.3%. The standard deviation of cell viabilities in the negative and the positive control substances, and the test substance were 3.7%, 0.2% and 5.8%, respectively. Since the acceptance criteria were met, the assay was considered valid.
The test substance was not observed to directly reduce MTT in the absence of viable cells.
Based upon the results of this assay, the test substance, L-Dihydrofarnesol, was identified as requiring classification and labelling according to UN GHS Category 1 or Category 2.

Applicant's summary and conclusion

Interpretation of results:

other: UN GHS Category 1 or Category 2

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

Based upon the results of this assay, the test substance L-Dihydrofarnesol, was identified as requiring classification and labelling according to UN GHS Category 1 or Category 2. Since the RhE test methods covered by TG439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion will be required to decide on its final classification.

Executive summary:

The MatTek Corporation’s EpiDerm™ reconstituted human epidermis model was used to assess the potential skin irritation of the test substance, L-Dihydrofarnesol, Lot Number 6K0001. The skin irritation potential of the test substance was evaluated by measuring the relative cell viability in treated tissues at 42-hour post-exposure incubation after a 60-minute exposure to the test substance, according to the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439). Under the conditions specified in this study, the test substance, L-Dihydrofarnesol, resulted in 41.2% cell viability and was predicted to be a skin irritant or corrosive. Results of the positive control and negative control met the criteria of a valid assay. Accordingly, the test substance, L-Dihydrofarnesol, would be identified as requiring classification and labelling according to UN GHS Category 1 or Category 2.