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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 16 December 2014 and 22 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it was conducted according to OECD Test Guideline 439 using a reconstructed human epidermis method and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
Identity: FRET 08-0338
Chemical name: 4,7-Methano-1H-inden-5-ol, octahydro-3,4,5-trimethyl and 4,7-Methano-1H-inden-5-ol, octahydro-2,4,5-trimethyl and isomers
CAS number: 1340502-69-3 & 1340502-93-3
Intended use: Fragrance ingredient in household products
Appearance: Clear liquid
Storage conditions: Room temperature (ca. 20˚C), in the dark

Test animals

Species:
other: Reconstructed human epidermis skin constructs
Strain:
other: Not applicable
Details on test animals and environmental conditions:
Receipt of tissues
On receipt, the kit contents were checked and the inserts with tissues on agarose were stored at room temperature until use. The kit was used within the expiry date indicated by the supplier (expiry date: 22 December 2014). The maintenance medium was pre-warmed to 37ºC. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.

Test system

Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
10 µL
Duration of treatment / exposure:
15 minutes
Observation period:
42 hours
Number of animals:
Not applicable
Details on study design:
Reduction of MTT by test substance
It is possible that a test substance may reduce MTT, resulting in the production of a blue colour without any involvement of cellular mitochondrial dehydrogenase. Since the test substance is rinsed off the tissue before the MTT assay, this is usually avoided. However, it is possible that small amounts of test substance may be present after washing or be released through the tissue into the MTT medium. If the mixture with the test substance turns blue/purple after approximately 3 hours incubation at 37 ± 2ºC in 5% CO2 in air, MTT reduction may have occurred.
The MTT reducing capability of the test substance, FRET 08-0338, was investigated by mixing 10 μL of the test substance with 2 mL of 0.3 mg/mL MTT solution in duplicate. A control of 10 μL of purified water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.

Check for colouring potential of test substance
The test substance, FRET 08-0338, was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). Evaluation was achieved by mixing 10 μL of the test substance, with 90 μL of purified water in a transparent container.
100 μL of purified water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the colour of the solution was assessed by eye.

Preparation/application of samples
The test substance, FRET 08-0338, a clear liquid, positive and negative controls were in liquid form and were applied by dispensing a volume of 10 µL over each tissue using a positive displacement pipette.

Test procedure
After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 ± 0.5 minutes with the test substance, negative or positive control at room temperature.
A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30 seconds and then re-spread with a curved flat spatula after 7 minutes application time.
After 15 ± 0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
After 42 ± 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
At the end of 3 hours ± 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro-tube.
When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration).
The tissues were extracted by storing at 2-8 ºC, protected from light, for 48 - 70 hours. After formazan extraction, duplicate 200 μL aliquots of the extractant from each tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Mean
Value:
9.6
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Remarks: Predicted as irritant to the skin. (migrated information)

In vivo

Irritant / corrosive response data:
It was concluded that the test substance, FRET 08-0338, with a mean tissue viability of 9.6 ± 3.1%, was predicted as irritant to the skin.

Any other information on results incl. tables

Check for colouring potential of test substance

The test substance, FRET 08-0338/water solution and water control were colourless after the 15 minute shaking period. The test substance had not shown any potential for colouring water.

 

Assay validity

Negative control

The mean absorbance of the triplicate negative control values was 0.884 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 14.3 which was below the maximum value of 18.

Positive control

The percentage mean viability of the positive control was 14.7 ± 1.1 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18%.

EpiSkin™ results

The results of the assay are summarised in the table below.

Sample

Tissue viability as percentage of mean OD negative control

Prediction MTT endpoint

Replicate tissues

Mean±SD

a

b

c

Negative control

88.

95.4

116.1

100.0±14.3

Not applicable

Positive control

16.0

14.0

14.3

14.7±1.1

Irritant

FRET 08-0338

12.8

6.6

9.4

9.6±3.1

Irritant

 

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It was concluded that the test substance, FRET 08-0338, with a mean tissue viability of 9.6 ± 3.1%, was predicted as irritant to the skin.
Executive summary:

The skin irritation potential of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 439 using a reconstructed human epidermis method. With a mean tissue viability of 9.6 ± 3.1%, the test substance was predicted as irritant to the skin.