Registration Dossier

Administrative data

Description of key information

Skin irritation:
In an in-vitro skin irritation study according to OECD 439 (Human Skin Model Test) 1,1-DPE was found to be irritative to the human skin.
Untreated DPE 75 (75% diphenyl ethane) was tested for oral and dermal toxicity in groups of 3 male rats. Skin irritation studies were performed with 3 New Zealand White rabbits in each case. A primary irritation index (PII) of 4 was obtained indicating that this material is a moderate skin irritant.
Untreated DPE75 is moderately irritating to the skin which is probably caused by a defatting action.
Eye irritation:
In-vivo eye irritation studies were performed with 3 New Zealand White rabbits in each case. Untreated DPE 75 (75% diphenyl ethane) was placed into the eye of each rabbit by gently pulling the lower lid away from the eyeball to form a cup. Assessment of ocular irritation was made 1, 24, 48 and 72-hours after treatment using the Draize Scoring system. According to the CLP and DSD criteria this substance was not considered to be irritant to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-08-22 - 2012-10-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The GLP study was conducted according to an internationally accepted guideline. All study parameters are given in detail.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: not applicable, human skin model
Strain:
other: not applicable, human skin model
Type of coverage:
other: not applicable, human skin model
Preparation of test site:
other: not applicable, human skin model
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
30 µl per tissue
Duration of treatment / exposure:
60 minutes
Details on study design:
P re-Tests

First, it was tested whether the test item develops a colour without MTT addition. 30pL were given in a test tube with 0.3 mL H20 demin. and incubated at 37 °C and 5 % C02 for 60 minutes. The resulting solution was colourless, therefore no binding capacity had to be tested.

Then, the test item (phenylethyi)benzene was tested for the ability of direct formazan reduction. To test for this ability, 30 pL were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 °C and 5 % CO2 for 60 minutes. Untreated MTT solution was used as control. The MTT solution didn't change its colour within one hour, therefore, direct MTT reduction had not taken place, and no data correction was necessary. Finally, (phenylethyl)benzene was tested for possible reaction with the nylon mesh which is used to ensure sufficient contact with the tissue surface. 30 |jL (phenylethyl)benzene were brought onto a nylon mesh on a microscope slide. No reaction with the mesh was visible after 1 h incubation at 37 °C.

Pre-lncubation of Tissues

Eight 6-well-plates were prepared with 0.9 mL assay medium in three of the six wells (upper row). The tissues were inspected for viability. Viable tissues were transferred (three per plate) in the wells with the medium using sterile forceps under the laminar air flow bank and placed into the incubator at 37 °C and 5% CO2 for one hour.

After the pre-incubation (one hour), the other three wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 °C and 5 % C02 for for 18 hours.

Treatment

The pre-incubated tissues were placed into fresh 6-weil-piates containing 0,9 mL assay medium per well, using the upper row only.

One plate (three tissues) was used as negative control; each tissue was treated with 30 pL DPBS buffer. One plate was used as positive control; each tissue was treated with 30 pL SDS-solution. One plate was used for treatment with the test item. 30 pL test item were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.


MTT Assay
After a total incubation time of 42 hours, a 24-well-plate was prepared with 300 pL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours.
After this time, the MTT reagent was aspirated and replaced by PBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipet¬ted, taking care to reach the upper rim of the insert. The plate was then shaked for two hours room temperature.
After two hours, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 pL solution (each) were pipetted into a 96-well- plate which was read in a plate spectral photometer at 570 nm.

Skin irritation potential of the test item is assessed as given in the following table:
% Formazan production Assessment
< 50% of negative control Irritant
> 50% of negative control Non-irritant
Irritation / corrosion parameter:
other:
Value:
33.8
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 min. Max. score: 0.0. Reversibility: no data. (migrated information)

%Formazan Production

Designation

(phenylethyl)benzene

Positive Control

% Formazan production (Tissue 1)

38.0 %

8.2 %

% Formazan production (Tissue 2)

34.0 %

7.7 %

% Formazan production (Tissue 3)

29.6 %

7.8 %

% Formazan production Mean

33.8 %

7.9 %

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item (phenylethyl)benzene is considered irritant.
Executive summary:

Three tissues of the human skin mode! EpiDerm™were treated with (phenylethyl)benzene for 60 minutes.

30 µL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size (0.63 cm2; as indicated by supplier).

DPBS-buffer was used as negative control, 5% SDS-solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable (<18 %).

After the treatment with the test item, the relative absorbance values were reduced to 33.8 %. This value is well below the threshold for irritation potential (50 %). Therefore, (phenylethyl)benzene is considered as "irritant in the Human Skin Model Test".
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No data gaps were identified. The available data are adequate for risk assessment and classification and labelling purposes.


Justification for selection of skin irritation / corrosion endpoint:
In an in-vitro skin irritation study according to OECD 439 (Human Skin Model Test) 1,1-DPE was found to be irritative to the human skin. The study is GLP-compliant and has Klimisch score 1.

The in vivo skin irritation potential of untreated 1,1-DPE was tested similar to OECD guideline 404. Skin irritation studies were performed with 3 New Zealand White rabbits in each case. A primary irritation index of 4 was obtained indicating that this material is a moderate skin irritant.
Since the in vitro testing gave positive results and other members of the category were tested positive for skin irritation, 1,1-DPE is considered as skin irritant, but not as skin corrosive as supported by the negative result in the in vivo assay.

Justification for selection of eye irritation endpoint:
In an in-vivo eye irritation study similar to OECD 405 untreated DPE 75 (75% diphenyl ethane) was tested negativ for eye irritation in the rabbit.The study is GLP-compliant and has Klimisch score 2.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

According to the CLP Regulation, the substance is classified as:

Skin Irritant, Category 2, based on Formazan production < 50% of the negative control.

Eye irritation: 1,1 -DPE is not classified as eye irritant.