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Description of key information

Skin corrosion (OECD TG 431): non-corrosive

Skin irritation (OECD TG 439):

The possible skin irritation potential of the substance was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439. Skin tissue was treated by topical application of 10 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 106.6%.

Since the mean relative tissue viability for FRET 11 -0539 was higher than 50% after 15 minutes treatment the substance is considered to be non irritant. The positive control had a mean cell viability of 8.2% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Eye severe damage (OECD TG 437): not classified


Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was conducted on 30 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 431 using the EPIDERM™ Reconstructed Human Epidermis Model and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item: FRET 11-0539
Test item identity (including alternative names): Benzene, 1-(2-methylpropyl)-4-(propoxymethyl)-; 4-(2-methylpropyl)benzyl propyl ether
Storage: conditions: Room temperature (15 - 25˚C)
Appearance: Clear, colorless liquid
Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: derived from human skin, number of donors not specified
Source strain:
other: derived from human skin
Justification for test system used:
The EpiDerm™ Human Skin Model for Skin Corrosivity Testing was endorsed by the European Centre for the Validation of Alternative Methods (ECVAM) on 20 March 2000.
Vehicle:
unchanged (no vehicle)
Details on test system:
The cultured tissues were supplied as kits by MatTek In Vitro Life Sciences Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovak Republic (Slovakia). The kits included assay medium, plates and the tissues which were shipped on agarose in sealed pouches.

Tissue Storage
On receipt, the contents of the kit were checked and the sealed pouch containing inserts of live tissues on agarose were stored refrigerated until use. The kit was used within the expiry date indicated by the supplier (expiry date: 01 December 2017).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): 100% (as supplied)

NEGATIVE CONTROL (purified water)
- Amount(s) applied (volume or weight): 50 µL


POSITIVE CONTROL (8.0N Potassium Hydroxide)
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8.0 N
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hrs
Number of replicates:
A total of 12 tissues were used: Duplicate tissues were treated with: test substance, positive control or negative control for each exposure time
Type of coverage:
other: substance was applied directly to the tissue
Preparation of test site:
other: The test item was applied topically to the corresponding tissues.
Vehicle:
unchanged (no vehicle)
Details on study design:
Reduction of MTT by Test Item
It is possible that a test item may reduce MTT, resulting in the production of a blue/purple colour without any involvement of cellular mitochondrial dehydrogenase. Since the test item is rinsed off the tissue before the MTT assay, this is usually avoided. However, it is possible that small amounts of test item may be present after washing or be released through the tissue into the MTT solution. If the mixture with the test item turned blue/purple after approximately one hour incubation in the dark at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, MTT reduction may have occurred.
The MTT reducing capability of the test item, FRET 11-0539, was investigated by mixing 50 µL of the test item with 1 mL of 1 mg/mL MTT solution in duplicate. A control of 1 mL of 1 mg/mL MTT solution was also included in duplicate.

Check for Colouring Potential of Test Item
The test item, FRET 11-0539, was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). Evaluation was achieved by mixing 50 µL of the test item, with 300 µL of purified water in a transparent micro-tube. 300 µL of purified water was included as a control. The solution was incubated at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air for one hour. At the end of the incubation period the colour of the solution was assessed by eye.

Preparation/Application of Samples
The controls and the test item, FRET 11-0539, were in liquid form. A volume of 50 µL was dispensed over each tissue using a suitable pipette.

Test Procedure
At least one hour before dosing, the tissues were transferred to 6-well plates containing 0.9 mL pre-warmed assay medium per well, then incubated at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
During the pre-incubation period, two sets of 24-well plates were prepared. The first set were holding plates containing 300 µL assay medium and the second set MTT assay plates containing 300 µL MTT medium (diluted MTT 1 mg/mL).
At least one hour after pre-incubation, the medium below the tissue inserts was replaced with 0.9 mL fresh assay medium. Plates awaiting sample application were returned to the incubator. Duplicate tissues were dosed for three minutes and duplicate tissues dosed for one hour with test item, negative control or positive control.
For practical purposes, dosing of the one hour tissues and the three minute tissues were performed as two separate batches. The one hour tissues were dosed first and returned to the incubator for the completion of the one hour dosing period. The three minute tissues were dosed during the one hour dosing period.
After the required dosing period, each tissue was rinsed with Dulbecco’s Phosphate Buffered Saline (DPBS), for a minimum of 20 rinses, and then blotted on absorbent paper. The inserts were then transferred to the holding plate. When all tissues from one batch had been transferred to a holding plate, the inserts were transferred to the MTT assay plate which was then incubated for three hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. After three hours, the tissues were rinsed with DPBS and transferred to 1 mL per well extraction solution (isopropanol) in 24-well plates. A further 1 mL was then added to each insert. The tissues were extracted for two hours with shaking at room temperature.
After the extraction period, triplicate 200 µL aliquots of extractant from each tissue were pipetted into the wells of flat-bottomed 96-well plates and the absorbance read at 540 nm.

Irritation / corrosion parameter:
other: relative mean viability (%)
Run / experiment:
3 minute exposure
Value:
95.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The test item was considered to be non-corrosive to the skin
Irritation / corrosion parameter:
other: relative mean viability (5%)
Run / experiment:
60 minute exposure
Value:
99.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The test item was considered to be non-corrosive to the skin.
Other effects / acceptance of results:
Possible Reduction of MTT by Test Item
There was no change in the test item, FRET 11-0539 /MTT solution or the water control/MTT solution (red/orange) after the one hour incubation in the dark at 37 ± 2ºC in a humidified atmosphere of 5% CO2 in air. The test item had not interacted with the MTT.

Check for Colouring Potential of Test Item
The test item, FRET 11-0539 /water solution and water control were colourless after the one hour incubation in the dark at 37 ± 2°C in a humidified atmosphere of CO2 in air. The test item had not shown any potential for colouring water.

Assay Validity
Negative Control
The mean absorbance of the duplicate negative control values for the three minute and one hour contact were 1.542 and 1.488, respectively. Both values were within the acceptance range of ≥ 0.8 to ≤ 2.8.
Positive Control
The mean relative tissue viability of the positive control, 8.0 N potassium hydroxide, for the one hour application was 2.0%. This value was below the maximum acceptable value of 15%.

Inter-Tissue Viability Difference
The percentage coefficient of variation (%CV) was not applicable for the positive control three minute and one hour applications as the mean percentage viabilities, 9.1% and 2.0% respectively, were outside the 20% - 100 % viability range. All other %CV values did not exceed 30%.

EpiDermResults

Sample

Tissue viability as % of mean negative control value

Prediction

3 minute contact

1 hour contact

Replicate

Mean±SD

Replicate

Mean±SD

FRET 11-0539

93.3

95.4± 3.057

103.0

99.2 ± 5.403

Non-

Corrosive

97.6

%CV = 3.204

95.4

%CV = 5.447

Negative Control

(Water)

107.3

100.0 ± 10.333

98.0

100.0 ± 2.789

Not

Applicable

92.7

%CV = 10.333

102.0

%CV = 2.789

Positive control

(8.0N KOH)

9.6

9.1 ± 0.703

2.0

2.0 ± 0.111

Corrosive

8.6

%CV = N/A

1.9

%CV = N/A

SD = Standard Deviation                  

%CV = Percentage Coefficient of Variation

N/A Not applicable

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item, FRET 11-0539, was classified as non-corrosive to the skin. The following classification criteria apply:
EU CLP (1272/2008/EC)/UN GHS: Not classified for corrosivity.
UN Packing Group: Non-Corrosive.
Executive summary:

The skin corrosivity of the test substance was determined according to OECD Guideline 431 using the EpiDerm™ Reconstructed Human Epidermis Model. The relative mean viability of 3 and 60 minute exposures were above 50 and 15% respectively in relation to the control. Therefore the substance is not corrosive to skin, according to EU CLP criteria.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

In vitro tests

Skin corrosion: The test item,FRET 11-0539, elicited a mean tissue viability of 95.4% for three minute contact and 99.2% for one hour contact and was predicted as non-corrosive in the EpiDerm skin corrosivity test.

Skin irritation: The skin irritancy of the test substance, FRET 11 -0539 was determined according to OECD Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model. The relative mean viability of the test item treated tissues was xx % after a 15-Minute exposureperiod and 42 hours post-exposure incubation period. This result shows that the substance is not a skin irritant, according to EU CLP criteria.

e non-irritant, according to EU CLP criterial. 


Justification for selection of skin irritation / corrosion endpoint:
The result of the study is reliable and adequate for covering the endpoint.

Effects on skin irritation/corrosion: non-corrosive, non-irritating

Eye irritation:

The eye irritancy potential of the test substance was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and is not classified, according to EU CLP criteria.


Justification for selection of eye irritation endpoint:
The result of the study is reliable and adequate for covering the endpoint.

Justification for classification or non-classification

Based on the negative results in the skin corrosion and skin irritation tests the substance does not need to be classified for this endpoint according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and according to EU Directive 67/548/EEC (DSD).

Based on the negative results in the eye irritation tests the substance does not need to be classified for this endpoint according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and according to EU Directive 67/548/EEC (DSD).